CN111587120A - Immunogenic compositions and uses thereof - Google Patents

Abstract

The present invention discloses the use of inhibitors of protein kinase C (PKC- θ) for enhancing the immune effector function of functionally inhibited T cells that have undergone epithelial to mesenchymal transition (EMT). In particular embodiments, PKC-theta inhibitors are disclosed for enhancing the sensitivity of exhausted T cells to reactivation of PD-1 binding antagonists. The compositions of the invention are useful for treating a range of disorders, including T cell dysfunctional disorders, such as pathogenic infections and hyperproliferative disorders.

Description

Immunogenic compositions and uses thereof

Technical Field

Priority of australian provisional application No. 2017904540 entitled "immunogenic composition and use thereof", filed 2017, 11, 8, the contents of which are incorporated herein by reference in their entirety.

The present invention relates generally to immunogenic compositions. More specifically, the present invention relates to the use of protein kinase C (PKC-theta) inhibitors for enhancing the immune effector function of functionally inhibited T cells that have undergone an epithelial to mesenchymal transition (EMT). In particular embodiments, PKC-theta inhibitors are used to enhance the sensitivity of exhausted T cells to reactivation of PD-1 binding antagonists. The compositions of the invention are useful for treating a range of disorders, including T cell dysfunctional disorders, such as pathogenic infections and hyperproliferative disorders.

Background

Programmed death receptor 1(PD-1) is an immune checkpoint modulator that can be expressed in a variety of immune cells, including T cells, B cells, Natural Killer (NK) cells, NK T (NKT) cells, monocytes, macrophages and Dendritic Cells (DCs) following their activation. PD-1 binds to two of its ligands: programmed cell death 1 ligand-1 (PD-L1; B7-H1; CD274) and PD-L2 (B7-DC; CD273), both of which are members of the B7 family. PD-L1 is constitutively expressed in a variety of cells including hematopoietic and non-hematopoietic cells. In contrast, expression of PD-L2 was restricted to specialized antigen presenting cells (APC; monocytes, macrophages and DCs) and to certain subsets of B cells. Inflammatory cytokines such as interferons (IFN; alpha, beta and gamma) are potent modulators of PD-L1 and PD-L2 expression.

PD-1 is induced by T Cell Receptor (TCR) signaling, which inhibits TCR/CD28 signaling and T cell activation when PD-1 binds to PD-LI or PD-L2. These immunomodulatory effects of PD-1 may limit T cell over-activation, thereby preventing immune-mediated tissue damage. However, prolonged TCR stimulation and PD-1 expression leads to T cell exhaustion, a state of T cell dysfunction defined by poor T cell effector function, sustained expression of inhibitory receptors, and transcriptional state distinct from functional effector or memory T cells, which is often associated with ineffective tumor control and persistent viral infection (Wherry, EJ.,2011.Nature Immunology 12: 492-499). Thus, the PD-1 pathway is an important determinant of T cell response outcome, which regulates the balance between effective host defense and immunopathology, suggesting the potential for manipulation of the PD-1 pathway against a variety of human diseases.

Blockade of the PD-1 pathway has been used to reactivate exhausted T cells and restore an anti-tumor or anti-pathogen immune response. Indeed, antibodies that block the PD-1 pathway have shown encouraging clinical outcomes in a significant number of patients with advanced cancer. However, clinical trial data to date have shown that the response rates of different types of cancer to PD-1 immune checkpoint inhibitory therapy vary widely, ranging from 18% to 87%. These trials also found that patients may exhibit primary, adaptive or even acquired tolerance to PD-1 immune checkpoint inhibitory therapies. Furthermore, emerging data indicate that some patients experience excessive progression of the disease state after receiving anti-PD-1 antibodies.

More recently, analysis of the immune profiles of peripheral blood of stage IV melanoma patients before and after treatment with anti-PD-1 antibody (pembrolizumab) by Huang et al (2017, Nature 545: 60-65) identified CD 8T cells (T cells) of the circulating exhaustion phenotype_exCells) in a subject. Most patients exhibit an immune response to pembrolizumab, but this is transient. In many patients, clinical failure is not only due to failure to induce immune reactivation, but also an imbalance between T cell reactivation and tumor burden. Circulating T measured in tumor burden before treatment_exThe magnitude of cellular reactivation is correlated with clinical response, which increases the likelihood that even a robust reactivation with anti-PD-1 therapy may be clinically ineffective if tumor burden is high.

Disclosure of Invention

The present invention results from the unexpected discovery that increasing protein kinase C theta (PKC-theta) in T cells (e.g., CD 8)⁺T cell) translocation in nucleusLeading to epithelial to mesenchymal transition (EMT) of cells, inhibiting their immune effector function, including expression of biomarkers that reduce T cell activation and effector capacity (e.g., interleukin 2(IL-2), interferon- γ (IFN- γ), and tumor necrosis factor- α (TNF- α)), and increasing expression of zinc finger E-Box binding homeobox 1(ZEB1), ZEB1 is a negative regulator of T cell response associated with cancer progression and inhibition of T cell effects, including inhibiting expression of IL-2 and E-cadherin, and inducing EMT.

The present inventors have also discovered that exposure of these mesenchymal, functionally inhibited T cells to PKC-theta inhibitors results in T cell epigenetic reprogramming and significantly relieves the suppression of their immune effector functions, including increased expression of biomarkers of T cell activation and effector capacity (e.g., IL-2, IFN-gamma, and TNF-alpha), decreased expression of biomarkers of T cell effector suppression and cancer progression (e.g., ZEB1), and decreased expression of biomarkers of T cell exhaustion (e.g., PD-1 and egiosodermin (EOMES)), and increased expression of the transcription factor TBET, which increases IFN-gamma production in cells of the adaptive and innate immune systems. Surprisingly, PKC-theta inhibitor-mediated epigenetic reprogramming has also been found to confer increased sensitivity to reactivation of PD-1 binding antagonists on exhausted T cells. These findings have been translated into the practice of methods and compositions for enhancing immune effector function of T cells and for treating diseases or disorders associated with T cell dysfunction, as described below.

Accordingly, in one aspect, the invention provides methods for enhancing T cells (e.g., CD 8)⁺T cell) function, or for use in treating a T cell dysfunctional disorder. These compositions generally comprise, consist of, or consist essentially of a PKC-theta inhibitor and a PD-1 binding antagonist. The PKC-theta inhibitor is suitably selected from inhibitors of PKC-theta enzymatic activity and inhibitors of PKC-theta nuclear translocation. In particular embodiments, PKC-theta is an inhibitor of PKC-theta nuclear translocation, non-limiting examples of which include peptides corresponding to the nuclear localization site of PKC-theta, such as those disclosed, for example, in International publication WO 2017/132728 Al (e.g., importib 4759). PD-1 binding antagonists suitably inhibit the binding of PD-1 to PD-L1 and/or PD-L2. In a preferred embodiment, the PD-1 binding antagonist is an anti-PD-1 antagonist antibody, illustrative examples of which include nivolumab (nivolumab), pembrolizumab (pembrolizumab), lambdavozolozumab (lambrolizumab), and pidilizumab (pidilizumab). In other embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., AMP-224). In some embodiments, the composition further comprises an adjuvant (e.g., a chemotherapeutic agent) for treating or aiding in the treatment of a T cell dysfunctional disorder.

Suitably, the enhanced T cell function includes any one or more of increased production of cytokines (e.g., IL-2, IFN- γ, TNF- α), increased CD8⁺T cell activation, increased T cell receptor recognition of antigens or antigenic peptides derived from antigens in the case of MHC class I molecules, increased clearance of cells presented by MHC class I molecules, and increased cytolytic killing of target cells expressing the antigen. In some embodiments, the T cell has a mesenchymal phenotype. Suitably, the T cell has aberrant expression of nuclear PKC-theta. In representative examples of this type, the T cell expresses nuclear PKC-theta at a level that is higher than the level of TBET expression in the same T cell, and/or at a level that is higher than the level of expression in an activated T cell. In certain of the same and other embodiments, the T cell is a T cell that exhibits T cell exhaustion or anergy. In a non-limiting example of this type,t cells express EOMES at levels higher than TBET and/or have increased PD-1 expression. Preferably, the T cell is CD8⁺T cells.

The present inventors propose that, since PKC-theta mediated EMT occurs in both tumor cells and T cells, regardless of cell type, PKC-theta mediated epigenetic reprogramming is likely to occur more broadly, including in other PD-1 expressing immune effector cells (e.g., T cells, B cells, NK cells, NKT cells, monocytes, macrophages, and DCs), thereby suppressing their immune effector function. Thus, in another aspect, the invention provides a method of enhancing the immune effector function of an immune effector cell expressing PD-1. These methods generally comprise, consist of, or consist essentially of contacting an immune effector cell with a PKC-theta inhibitor and a PD-1 binding antagonist, thereby enhancing an immune effector function of the immune effector cell. Suitably, the enhanced immune effector function comprises any one or more of: increased recognition of antigens or antigenic peptides by T cell receptors (said antigens or antigenic peptides being derived from antigens in the case of MHC class II molecules), increased cytokine release and/or CD8⁺In a representative example of this type, the immune effector cells express nuclear PKC-theta at levels greater than expression levels of control immune effector cells (e.g., immune effector cells having normal or non-suppressed immune effector function).

In another aspect, the invention provides methods of treating T cell dysfunction in a subjectMethods of treating sexual disorders. These methods generally comprise, consist of, or consist essentially of simultaneously administering to the subject an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist to treat a T cell dysfunctional disorder. Suitably, the PKC-theta inhibitor and the PD-1 binding antagonist are administered in synergistically effective amounts. In some embodiments, the T cell dysfunctional disorder is a disorder or condition of T cells characterized by decreased response to antigen stimulation and/or increased inhibitory signaling by PD-1. In some of the same and other embodiments, the T cell dysfunctional disorder is one in which the T cell has a reduced ability to secrete cytokines, proliferate, or undergo cytolytic activity. In this type of illustrative example, a reduced response to antigenic stimulation results in ineffective control of the pathogen or tumor. In some embodiments, the T cell dysfunctional disorder is one in which T cells are anergic. Representative examples of T cell dysfunctional disorders include unresolved acute infections, chronic infections, and tumor immunity. In a preferred embodiment, the T cell dysfunctional disorder is a cancer or infection comprising T cells having a mesenchymal phenotype (e.g., CD 8)⁺T cells). In representative examples of this type, the T cell expresses nuclear PKC-theta at a level that is higher than the level of TBET expression in the same T cell, and/or at a level that is higher than the level of expression in an activated T cell. In some of the same and other embodiments, the T cell is a T cell that exhibits T cell fatigue or anergy. In non-limiting examples of this type, the T cells express EOMES at a level greater than the level of TBET and/or have increased PD-1 expression. In some embodiments, the T cell is a tumor infiltrating lymphocyte. In other embodiments, the T cell is a circulating lymphocyte. In some embodiments, the cancer is skin cancer (e.g., melanoma), lung cancer, breast cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, renal cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, orHepatocellular carcinoma. In a preferred embodiment, the cancer is a metastatic cancer. Preferably, the metastatic cancer is metastatic melanoma or metastatic lung cancer. In some embodiments, the methods further comprise administering to the subject a PKC-theta inhibitor and a PD-1 binding antagonist, adjuvant (e.g., chemotherapeutic agent), or adjuvant therapy (e.g., ablative or cytotoxic therapy) simultaneously for treating or adjunctively treating the T cell dysfunctional disorder.

In a related aspect, the invention provides methods of treating or delaying progression of cancer in a subject. These methods generally comprise, consist of, or consist essentially of administering to the subject an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist simultaneously to treat or delay progression of cancer. In some embodiments, the subject has been diagnosed with cancer, wherein in a tumor sample of the cancer from the subject, the T cells express nuclear PKC-theta at a level that is higher than the level of expression of TBET in the same T cells, and/or higher than the level of expression in activated T cells.

In other related aspects, the invention provides methods of enhancing immune function (e.g., immune effector function) in an individual having cancer. These methods generally comprise, consist of, or consist essentially of, administering to the individual an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist simultaneously to the individual to enhance immune function. In some embodiments, an individual has been diagnosed with cancer, wherein a tumor sample of the cancer taken from the individual expresses nuclear PKC-theta at a level that is greater than the level of expression of TBET in the same T cell, and/or greater than the level of expression in an activated T cell.

In other aspects, provided herein are methods of treating an infection (e.g., a bacterial or viral or other pathogen infection). The methods generally include, consist of, or consist essentially of simultaneously administering to the individual an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist to treat the infection. In some embodiments, the infection is a viral and/or bacterial infection. In some embodiments, the infection is a pathogen infection. In some embodiments, the infection is an acute infection. In some embodiments, the infection is a chronic infection.

In other related aspects, the invention provides methods of enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having an infection. These methods generally comprise, consist of, or consist essentially of, administering to the individual an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist simultaneously to the individual to enhance immune function. In some embodiments, the individual has been diagnosed with an infection, wherein in a sample from the individual, the T cells express nuclear PKC-theta at a level that is greater than the level of expression of TBET in the same T cells, and/or greater than the level of expression in activated T cells.

Another aspect of the invention provides the use of a PKC-theta inhibitor and a PD-1 binding antagonist for the treatment of a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection. PKC-theta inhibitors and PD-1 binding antagonists are commonly used in the preparation of medicaments for this purpose. Suitably, the PKC-theta inhibitor and the PD-1 binding antagonist are formulated for simultaneous administration.

In related aspects, the invention provides for the use of PKC-theta inhibitors, PD-1 binding antagonists and adjunctive agents (e.g., chemotherapeutic agents) for the treatment or for the adjunctive treatment of T cell dysfunctional disorders, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in individuals with cancer, for treating or delaying progression of cancer, or for treating infection. PKC-theta inhibitors, PD-1 binding antagonists, and adjuvants (e.g., chemotherapeutic agents) are commonly used in the preparation of medicaments for this purpose. Suitably, the PKC-theta inhibitor, PD-1 binding antagonist, and adjuvant (e.g., chemotherapeutic agent) are formulated for simultaneous administration.

In some embodiments, a method for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection, comprises detecting elevated nuclear PKC-theta (i.e., PKC-theta localized in the nucleus) levels (e.g., relative to TBET levels in the same T cell, or nuclear PKC-theta levels in an activated T cell) in T cells in a sample obtained from a subject prior to concurrent administration.

In some embodiments, a method for treating a T cell dysfunctional disorder comprises detecting, prior to the concurrent administration, elevated nuclear PKC-theta (i.e., PKC-theta localized in the nucleus) levels (e.g., relative to TBET levels in the same T cell, or nuclear PKC-theta levels in an activated T cell) in T cells in a sample obtained from the subject, and elevated ZEB1 levels in the nucleus of the T cells (e.g., relative to TBET levels in the same T cell, or ZEB1 levels in the nucleus of the activated T cell). In representative examples of this type, the methods include detecting an elevated level of a complex comprising PKC-theta and ZEB1, as appropriate, in the nucleus of a T cell.

In a related aspect, the invention provides a kit comprising a medicament comprising a PKC-theta inhibitor and optionally a pharmaceutically acceptable carrier, and a package insert comprising instructional material for simultaneously administering the medicament and another medicament comprising a PD-1 binding antagonist and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying the progression of cancer, or for treating an infection in an individual.

In other related aspects, the invention provides kits comprising a medicament comprising a PD-1 binding antagonist and optionally a pharmaceutically acceptable carrier, and a package insert comprising instructional material for simultaneously administering the medicament and another medicament comprising a PKC-theta inhibitor and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying the progression of cancer, or for treating an infection in an individual.

In other related aspects, the invention provides kits comprising a first medicament comprising a PKC-theta inhibitor and optionally a pharmaceutically acceptable carrier, and a second medicament comprising a PD-1 binding antagonist and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection in an individual. In some embodiments, the kit further comprises a package insert comprising instructional material for simultaneously administering the first drug and the second drug for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection in an individual.

In some embodiments of the methods, uses, compositions, formulations, and kits described above and elsewhere herein, CD8 in the individual is compared to prior to administering the combination of the PKC-theta inhibitor and the PD-1 binding antagonist⁺T cells have enhanced priming, activation, proliferation and/or cytolytic activity. In some embodiments, CD8 compared to prior to administration of the combination⁺The number of T cells increases. In some embodiments, CD8⁺T cells are antigen-specific CD8⁺T cells. In some embodiments, the administration of a PKC-theta inhibitor andin some embodiments, the number of Treg cells is reduced as compared to prior to administration of a combination of a PKC-theta inhibitor and a PD-1 binding antagonist, in some embodiments, plasma IFN- γ is increased as compared to prior to administration of a combination of a PKC-theta inhibitor and a PD-1 binding antagonist, in some embodiments, plasma TNF- α is increased as compared to prior to administration of a combination of a PKC-theta inhibitor and a PD-1 binding antagonist, in some embodiments, plasma IL-2 is increased as compared to prior to administration of a combination of a PKC-theta inhibitor and a PD-1 binding antagonist.

In some embodiments of the methods, uses, formulations, and kits described above and elsewhere herein, the PKC-theta inhibitor and/or PD-1 binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implant, inhalations, intrathecally, intraventricularly, or intranasally. In some embodiments of the methods, uses, compositions, and kits described above and herein, the treatment further comprises administering an adjuvant (e.g., a chemotherapeutic agent) for treating or delaying the progression of the cancer in the individual. In some embodiments, the subject has been treated with a chemotherapeutic agent prior to treatment with the combination of a PKC-theta inhibitor and a PD-1 binding antagonist. In some embodiments, the treated subject is refractory to treatment with a chemotherapeutic agent. Some embodiments of the methods, uses, compositions, and kits described throughout the application further comprise administering a chemotherapeutic agent for treating or delaying the progression of cancer.

Another aspect of the invention provides a method of diagnosing the presence of a T cell dysfunctional disorder in a subject. These methods generally comprise, consist of, or consist essentially of the steps of:

(i) obtaining a sample from a subject, wherein the sample comprises T cells (e.g., CD 8)⁺T cells);

(ii) contacting the sample with a first binding agent that binds PKC-theta in the sample and a second binding agent that binds ZEB1 in the sample; and

(iii) detecting the localization of the first binding reagent and the second binding reagent in the nucleus of the T cell;

wherein localization of the first binding agent and the second binding agent in the nucleus of the T cell is indicative of the presence of a T cell dysfunctional disorder in the subject.

In another aspect, the invention provides a method of diagnosing the presence of a T cell dysfunctional disorder in a subject. These methods generally comprise, consist of, or consist essentially of the steps of:

(i) obtaining a sample from a subject, wherein the sample comprises T cells (e.g., CD 8)⁺T cells);

(ii) contacting the sample with a first binding agent that binds PKC-theta in the sample and a second binding agent that binds ZEB1 in the sample; and

(iii) detecting the first binding agent and the second binding agent when bound to PKC-theta-ZEB 1 complex in the sample;

wherein an increase in the level of PKC-theta-ZEB 1 complex detected in the sample relative to the level of PKC-theta-ZEB 1 complex detected in a control sample (e.g., a sample comprising activated T cells) is indicative of the presence of a T cell dysfunctional disorder in the subject.

Another aspect of the invention provides methods of monitoring treatment of a subject having a T cell dysfunctional disorder. These methods generally comprise, consist of, or consist essentially of the steps of:

(i) obtaining a sample from a subject following treatment of the subject with a therapy for a T cell dysfunctional disorder, wherein the sample comprises T cellsCell (e.g., CD 8)⁺T cells);

(ii) contacting the sample with a first binding agent that binds PKC-theta in the sample and a second binding agent that binds ZEB1 in the sample; and

(iii) detecting the first binding agent and the second binding agent when bound to PKC-theta-ZEB 1 complex in the sample;

wherein a lower level of PKC-theta-ZEB 1 complex detected in the sample relative to the level of PKC-theta-ZEB 1 complex detected in a control sample obtained from the subject prior to treatment indicates an increased clinical benefit (e.g., enhanced immune effector function, e.g., enhanced T cell function) in the subject, and

wherein a higher level of PKC-theta-ZEB 1 complex detected in the sample relative to the level of PKC-theta-ZEB 1 complex detected in a control sample obtained from the subject prior to treatment indicates no or negligible clinical benefit to the subject (e.g., enhanced immune effector function, e.g., enhanced T cell function).

In another aspect, a kit for diagnosing the presence of a T cell dysfunctional disorder in a subject is provided. These kits typically comprise, consist of, or consist essentially of: (i) a first binding agent that binds to PKC-theta, (ii) a second binding agent that binds to ZEB 1; and (iii) a third agent comprising a label (label) that is detectable when the first binding agent and the second binding agent each bind to a PKC-theta-ZEB 1 complex. In specific embodiments, the third agent is a binding agent that binds to the first and second binding agents.

In a related aspect, the present invention provides a complex comprising PKC-theta and ZEB1, a first binding agent that binds PKC-theta of said complex, a second binding agent that binds ZEB1 of said complex; and (iii) a third agent comprising a label that is detectable when the first binding agent and the second binding agent each bind to a PKC-theta-ZEB 1 complex. In a specific embodiment, the PKC-theta-ZEB 1 complex is localized in a T cell. In specific embodiments, the third agent is a binding agent that binds to the first and second binding agents.

In another aspect, the present invention provides a T cell comprising a complex comprising PKC-theta and ZEB1, a first binding agent that binds PKC-theta of said complex, a second binding agent that binds ZEB1 of said complex; and (iii) a third agent comprising a label that is detectable when the first binding agent and the second binding agent each bind to a PKC-theta-ZEB 1 complex. In specific embodiments, the third agent is a binding agent that binds to the first and second binding agents.

In any of the above aspects, each binding agent is preferably an antibody.

The above diagnostic methods and kits can be used as companion diagnostic agents for the therapeutic methods of the present invention.

Drawings

Figure 1 is a representative graph, schematic and photograph showing that PKC-theta can be targeted for therapeutic intervention. A) The use of a probe such as Wagstaff et al (2011.Journal of Biomolecular Screening 16 (2)): 192-

Binding assay, recombinant purified His₆Binding strength of PKC-theta to increasing concentrations of different subunit and subunit combinations of the import protein (immunoprotein) α/β heterodimer nuclear transport receptor (α 2, α 2 β 1 and β 1) B-C) describes the protein structure of PKC-theta, indicating the binding localization to PKC-theta peptide inhibitors (PKC theta i (RKEIDPPFRPKVK)), including the Nuclear Localization Sequence (NLS) D) of PKC-theta detecting the specificity of PKC theta i on cells treated with this peptide inhibitor, screening of cells with a first antibody against PKC-theta (T538p), PKC- β 2, PKC- β 1, PKC- α, PKC-and PKC-gamma the ratio of nuclear to cytoplasmic fluorescence (Fn/C) using the equation Fn/C ═ Fb)/(Fn-Fb where Fn is nuclear fluorescence, Fc is cytoplasmic fluorescence, Fb is background fluorescence, and the effect of the nuclear translocation inhibitor on the background localization of PKC-theta (WSi) on the nuclear translocation of PKC-theta peptide inhibitors (WSi) was determined using the Sigma-theta peptide assay₅₀. Calculation of EC for inhibitors Using GraphPad PRISM software⁵⁰. F) Measuring PKC-theta kinase activity using a PKC kinase activity kit and recombinant PKC-theta purchased from ENZO life sciences; c27 is an inhibitor of PKC-theta kinase activity, disclosed in Jimenez et al (2013.J Med Chem 56 (5): 1799-.

FIG. 2 is a graph depicting CD8 from BRAF negative melanoma patients⁺The present invention relates to a method for screening for primary tumor resistance in melanoma patients, comprising the steps of (a) identifying a PKC-theta resistance signature (signature) in T cells, a representation of photographs and graphs of PKC-theta resistance signature (signature) in melanoma patients from baseline biopsy of primary tumors, using Leica Bond RX Stainer, treatment by 3D high resolution microscopy, fixing FFPE tissue and immunofluorescence microscopy with primary antibodies against PD1, PKC-theta (T538p) and CD8 and DAPI, (B) displaying representative images of each data set (C — C) (C) identifying peripheral blood mononuclear cells isolated from melanoma patients fluid biopsies, using ImageJ to measure TCFI values after background subtraction of CD8, tnpi of PKC-theta, and TCFI of PD1 (N ═ 40 cells per patient sample), B) (PBMC) isolated from melanoma patients, and (C — C) (C — C) identifying a primary tumor resistance signature) in melanoma patients, using ifc-theta peptide inhibitors, and (C — C) identifying peripheral blood mononuclear cells isolated from melanoma patients from PBMC fluid biopsies (C) by clinical screening, PBMC) using control or anti-C — C, (C) and anti-theta peptide inhibitors, CD-PD) (B) identifying a high resolution microscopy, anti-PD) to express primary tumor cells, anti-CD-PD, (C) after clinical screening, PBMC) and anti-PD) to express a high resolution microscopy, a contrast medium.

FIG. 3 is a diagram illustrating CD8⁺Photo representation of PKC-theta tolerance signature in T cells. a-B) CD8 isolated from liquid biopsies of melanoma patients (CR ═ complete response, SD ═ stable disease, PD ═ progressive disease), stimulated with PMA/CI and preclinical screening with vehicle controls or PKC θ i nuclear peptide inhibitors. The sample is fixed and the sample is fixed,representative images for each data set are shown representing TNFI for PKC-theta and TNFI for ZEB1 measured using ImageJ, nuclei (n ═ 20 cells/sample) after background subtraction are selected, maps for ZEB1 and PKC-theta are also shown for each group (red ═ ZEB1, green ═ PKC-theta) and pcc. maps depict the percent inhibition or induction based on protein expression, where C-D is also plotted against each protein target relative to untreated samples, CD8 isolated from liquid biopsy tissue of melanoma patients (CR ═ complete response, PR ═ partial response, PD ═ progressive disease), stimulated with PMA, and mapped against vehicle control or PKC θ i nuclear peptide inhibitors, fixed samples are screened preclinically using TNF-theta nuclear peptide inhibitors, and IFN-gamma-inhibition is also plotted against these samples, IFN-gamma-induced data are shown on IFN-dry basis of IFN-gamma-induction maps, where IFN-gamma-inhibition is plotted against IFN-dry tissue, and IFN-gamma-induced protein expression is also plotted against each protein target.

FIG. 4 is a graph showing CD8 of EOMES, TBET and PD-1 in melanoma patients fluid biopsies in the presence and absence of PKC θ i peptide inhibition⁺Representative illustration of expression in T cells. A) CD8 isolated from liquid biopsy of melanoma patients⁺T cells (CR ═ complete response, SD ═ stable disease, PD ═ progressive disease), stimulated with PMA/CI and preclinical screening with vehicle controls or PKC θ i peptide inhibitors. The samples were then fixed and these cells were subjected to immunofluorescence microscopy with primary antibodies against EOMES, TBET and PD-1. A representative image of each data set is displayed. Figure shows TCFI of PD1, TNFI of EOMES and TNFI of TBET measured using ImageJ (n ═ 20 cells/sample), and nuclei were selected with background subtracted. The graph depicts the percent inhibition or induction based on protein expression, wherein each protein target is also plotted against untreated samples.

Some graphics and text contain color representations or entities. The color illustrations may be obtained by request from the applicant or from an appropriate patent office. If obtained from the patent office, a fee may be charged.

Detailed Description

1. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. For the purposes of the present invention, the following terms are defined below.

The articles "a" (a) and "an" (an) as used herein refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" refers to one or more than one element.

As used herein, the term "about" refers to the usual error range for various values as would be readily understood by one of skill in the art. References herein to "about" a value or parameter include (and describe) embodiments that are directed to that value or parameter itself.

As used herein, "activation" refers to a cellular state after sufficient cell surface moieties are attached to induce a significant biochemical or morphological change. In the context of T cells, such activation refers to the state of the T cell that has been sufficiently stimulated to induce cell proliferation. Activation of T cells may also induce cytokine production and the performance of detectable effector functions, including regulatory or cytolytic effector functions. In the context of other cells, the term relates to the up-or down-regulation of a particular physicochemical process. Activation may also be associated with induced cytokine production and detectable effector function.

The term "activated T cell" refers to a T cell that is currently undergoing cell division, detectable effector function (including the performance of cytokine production, regulatory or cytolytic effector function), and/or has recently undergone an "activation" process.

The terms "simultaneous administration" or "co-administration" and the like refer to the administration of a single composition comprising two or more active substances, or the administration of each active substance as separate compositions and/or the simultaneous (simultaneous) or simultaneous (simultaneously) or sequential delivery by separate routes in a sufficiently short time that the effective results are equivalent to the results obtained when all of the active substances are administered as a single composition. By "simultaneous" is meant that the active agents are administered together at substantially the same time, and desirably in the same formulation. By "contemporaneous" is meant that the active agents are administered within a tight time period, e.g., within about one minute to about one day before or after one agent is administered to another agent. Any contemporaneous time may be used. However, it is often the case that when not administered simultaneously, the agents will be administered within about one minute to about eight hours, and suitably within less than about one hour to about four hours. When administered contemporaneously, the agents are suitably administered at the same site of the subject. The term "same site" includes a precise location, but may be within about 0.5 to about 15 centimeters, preferably within about 0.5 to about 5 centimeters. As used herein, the term "separately" refers to the administration of agents at intervals, for example, at intervals of about one day to several weeks or months. The active agents may be administered in either order. The term "sequentially" as used herein means that the agents are administered sequentially, e.g., at intervals of minutes, hours, days, or weeks. If appropriate, the active agent may be administered in a regularly repeated cycle.

The term "agent" includes compounds that induce a desired pharmacological and/or physiological effect. The term also encompasses pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, and the like. When the above terms are used, it is understood that this includes the active agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs and the like. The term "agent" should not be construed narrowly, but extends to small molecules, protein molecules, such as peptides, polypeptides and proteins, and compositions comprising the same, as well as genetic molecules, such as RNA, DNA and mimetics and chemical analogues thereof, and cellular agents. The term "agent" includes cells that are capable of producing and secreting a polypeptide referred to herein, as well as polynucleotides comprising a nucleotide sequence encoding the polypeptide. Thus, the term "agent" extends to nucleic acid constructs, including vectors such as viral or non-viral vectors, expression vectors, and plasmids for expression and secretion in a variety of cells.

As used herein, "amplification" generally refers to the process of producing multiple copies of a desired sequence. "multiple copies" means at least two copies. "copy" does not necessarily mean complete sequence complementarity or identity to the template sequence. For example, the copies may include nucleotide analogs, such as deoxyinosine, intentional sequence alterations (e.g., sequence alterations introduced by primers comprising sequences that are hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.

The "amount" or "level" of a biomarker is a level detectable in a sample. These can be measured by methods known to those skilled in the art and disclosed herein. The level or amount of expression of the biomarker assessed can be used to determine a response to treatment.

As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

The term "non-responsive" refers to an incomplete or inadequate signal due to delivery through a T cell receptor (e.g., increased intracellular Ca in the absence of ras activation²⁺) Resulting in a non-responsive state to the antigenic stimulus. Stimulation with antigen without co-stimulation can also result in T cells unresponsive, making it difficult for cells to be activated by subsequent antigen even with co-stimulation. The presence of IL-2 may generally cause the non-responsive state to be overridden. The non-responsive T cells do not undergo clonal expansion and/or gain effector function.

The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, inhibits, neutralizes, or reduces the biological activity or action of another molecule, such as a receptor.

The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effects of the target. In some embodiments, the term may refer to an antibody that prevents a target (e.g., PD-1) bound thereto from performing a biological function.

As used herein, an "anti-PD-1 antagonist antibody" refers to an antibody that is capable of inhibiting PD-1 biological activity and/or downstream events mediated by PD-1. anti-PD-1 antagonist antibodies include antibodies that block, antagonize, inhibit, or reduce (to any extent, including significantly) PD-1 biological activity, including inhibitory signal transduction by PD-1 and downstream events mediated by PD-1, such as PD-L1 binding and downstream signaling, PD-L2 binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and inhibition of anti-tumor immune response. For the purposes of the present invention, it will be clearly understood that the term "anti-PD-1 antagonist antibody" (interchangeably referred to as "antagonist PD-1 antibody", "antagonist anti-PD-1 antibody" or "PD-1 antagonist antibody") encompasses all previously identified terms, titles, functional states and characteristics in which PD-1 itself, the biological activity of PD-1 or consequences of the biological activity are substantially eliminated, reduced or neutralized to any meaningful degree. In some embodiments, the anti-PD-1 antagonist antibody binds to PD-1 and upregulates an anti-tumor or anti-pathogen immune response. Examples of anti-PD-1 antagonist antibodies are provided herein.

The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" antibody is one that has been identified and isolated and/or recovered from a component of its natural environment. Contaminant components in their natural environment are substances that would interfere with antibody research, diagnostic or therapeutic uses, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified to (1) greater than 95% by weight of the antibody, as determined by the Lowry method, in some embodiments, to greater than 99% by weight; (2) to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using, for example, a rotary cup sequencer, or (3) to achieve homogeneous conditions by SDS-PAGE under reducing or non-reducing conditions, for example, using coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment is not present. However, an isolated antibody will typically be prepared by at least one purification step.

A "natural antibody" is typically a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable domain (V)_H) Followed by a plurality of constant domains. Each light chain has a variable domain (V) at one end_L) And the other end has a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues will form an interface between the light and heavy chain variable domains.

The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of an immunoglobulin (i.e., a variable domain that comprises an antigen binding site). Constant Domain comprising heavy chain C_H1、C_H2And C_H3Domains (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of a heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "V_H". Light chain variableThe domains may be referred to as "V_L". These domains are usually the most variable parts of an antibody and contain an antigen binding site.

The term "variable" refers to the fact that certain portions of the variable domains differ greatly in sequence between antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed among the variable domains of the antibody. It is concentrated in three segments called hypervariable regions (HVRs) of the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, predominantly in a β -sheet configuration, and are connected by three HVRs, which form connecting loops and, in some cases, parts of the β -sheet structure. The HVRs in each chain are maintained in close proximity by the FR region and form the antigen-binding site of an antibody with HVRs from the other chain (see Kabat et al, Sequences of proteins of Immunological Interest, 5 th edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

The "light chains" of antibodies (immunoglobulins) from any mammalian species can be classified into one of two distinctly different types, called kappa (κ) and lambda (λ), respectively, based on the amino acid sequences of their constant domains.

The term IgG "isotype" or "subclass" as used herein refers to any subclass of immunoglobulin defined by the chemical and antigenic properties of its constant regions.

Antibodies (immunoglobulins) can be classified into different classes according to the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. Corresponding to different classes of immunoglobulinsThe subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al, Cellular and mol.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in substantially intact form, rather than to antibody fragments as defined below. The term particularly refers to antibodies having a heavy chain comprising an Fc region.

The term "naive T cell" refers to an immune cell comprising cells that do not encounter an antigen, e.g., an immune cell that is a precursor to a memory T effector cell. In some embodiments, naive T cells can be differentiated, but have not yet encountered their cognate antigen, and are therefore activated T cells or memory effector T cells. In some embodiments, the naive T cells are characterized by expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127, and the absence of CD95 or CD45RO isoforms.

For purposes herein, a "naked antibody" is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, an antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site and a residual "Fc" fragment, the name reflecting its ability to crystallize readily. Pepsin treatment to yield F (ab')₂A fragment having two antigen binding sites and still being capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In single chain Fv (scFv) classes, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker, enabling the light and heavy chains to bind in a "dimerized" structure similar to that of a two-chain Fv class. In this configuration, the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, six HVRs confer antibody antigen-binding specificity. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with less affinity than the entire binding site.

Fab fragments comprise both heavy and light chain variable domains, and also comprise the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the name for Fab' herein, in which one or more cysteine residues of the constant domain carry a free thiol group. F (ab')₂Antibody fragments were originally produced as a Fab' fragment pair with a hinge cysteine between them. Other chemical couplings of antibody fragments are also known.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the structure required for antigen binding. For reviews on scFv see, for example, Pluckthun, The pharmacy of monoclonal Antibodies, 113, Vol.Rosenburg and Moore eds (Springer-Verlag, New York,1994), p.269-315.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using linkers that are too short to allow pairing between two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Diabodies may be bivalent or bispecific. Diabodies are more fully described, for example, in P404,097; WO 1993/01161; hudson et al, nat. med.9: 129-134 (2003); and Hollinger et al, proc.natl.acad.sci.usa 90: 6444-6448(1993). Triabodies and tetrabodies are described in Hudson et al, nat. med.9: 129-134(2003) are also described.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the population comprises individual antibodies that are identical except for possible mutations, e.g., naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates that the antibody is not characterized as a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds a target, wherein the polypeptide sequence that binds a target is obtained by a method comprising selecting a single polypeptide sequence that binds a target from a plurality of polypeptide sequences. For example, the selection method can be to select a unique clone from a plurality of clones (e.g., a collection of hybridoma clones, phage clones, or recombinant DNA clones). It will be appreciated that the selected target-binding sequence may be further altered, for example, to increase affinity for the target, to humanize the target-binding sequence, to improve its yield in cell culture, to reduce its immunogenicity in vivo, to produce a multispecific antibody, etc., and that an antibody comprising the altered target-binding sequence is also a monoclonal antibody of the invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal Antibodies used according to the invention can be prepared by a variety of techniques, including, for example, the Hybridoma method (e.g., Kohler and Milstein, Nature, 256: 495-97 (1975); Hongo et al, Hybridoma,14 (3): 253- -260(1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2 nd edition 1988); Hammerling et al, monoclonal Antibodies and T-cell hybrids 563; Elsevier, N.Y.,1981), the recombinant DNA method (see, for example, U.S. Pat. No. 4,816,567), the phage display technique (see, for example, 2004, Nature, 352: Im 624-; Marks et al, J.222: 1247; Biodhl et al, 2000: 2000; Legend et al; Legend No. 3: 310; Legend et al; Legend, 120. 31; Legend, 76; Legend et al; Legend, 120. 76; Legend, 76; U.32; 35; Legend, D.32; U.32; Legend, D.),697; Legend et al; Legend, 120; U.310; Legend et al; 35; U.32; Legend; SEQ ID.),220; Legend et al; 35; Legend; U.32; U.3; U.32; Legend; SEQ ID; 35, and techniques for producing human or human-like antibodies in animals having a human immunoglobulin locus or gene encoding partially or wholly a human immunoglobulin sequence (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al, Nature 362: 255-258 (1993); Bruggemann et al, Yeast in Immunol. 7: 33 (1993); U.S. Pat. No. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S. Pat. No. 5,661,016; Marks et al, Bio/Technology 10: 779-783 (1992); Lonberg et al, Nature 368: 856-859 (1994); Morrison: Nature: Huuh H. 126; Nature: 368; Nature: H. technol. 1996; Nature: 14: 1996; Nature: 14: 93; Biosge., Nature: 1996; and Nature: 92: Nature 11: 93; Bioscher et al, Nature: 92).

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Mor;)rison et al, proc.natl.acad.sci.usa 81: 6851-6855(1984)). Chimeric antibodies include

An antibody, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, cynomolgus monkey immunized with an antigen of interest.

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not present in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance. Typically, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see, e.g., Jones et al, Nature 321: 522-525 (1986); riechmann et al, Nature 332: 323-329 (1988); and Presta, curr. op. struct.biol.2: 593-596(1992). See also, for example, Vaswani and Hamilton, ann. 105-115 (1998); harris, biochem. soc. transactions 23: 1035-; hurle and Gross, curr.op.biotech.5: 428-; and U.S. patent nos. 6,982,321 and 7,087,409.

A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human, and/or the amino acid sequence of an antibody that has been made using any of the techniques disclosed herein for making human antibodies. The personThe definition of antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies, including phage display libraries, can be produced using a variety of techniques known in the art. Hoogenboom and Winter, j.mol.biol., 227: 381 (1991); marks et al, j.mol.biol., 222: 581(1991). Methods for preparing human Monoclonal Antibodies can also be used, and are described in Cole et al, Monoclonal Antibodies and Cancer Therapy, AlanR.Liss, p.77 (1985); boerner et al, j.immunol.,147 (1): 86-95(1991). See also van Dijk and van de Winkel, curr, opin, pharmacol, 5: 368-74(2001). Human antibodies can be made by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has been inactivated, e.g., an immunized xenomic (see, e.g., U.S. Pat. nos. 6,075,181 and 6,150,584, which relates to xenomine @)^TMA technique). See also, for example, Li et al, proc.natl.acad.sci.usa, 103: 3557-3562(2006), which relates to human antibodies produced by human B cell hybridoma technology.

A "species-dependent antibody" is an antibody that has a higher binding affinity for an antigen from a first mammalian species than for an antigen homolog from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (e.g., has a binding affinity (Kd) value of no greater than about 1x10^-7M, preferably not greater than about 1x10^-8M, and preferably no greater than about 1x10^-9M), but has a binding affinity for a homolog of an antigen from a second non-human mammalian species that is at least about 50-fold weaker than its binding affinity for a human antigen, or at least about 500-fold weaker than its binding affinity for a human antigen. The species-dependent antibody may be any of the various types of antibodies as defined above, but is preferably a humanized or human antibody.

As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain which is hypervariable in sequence and/or forms structurally defined loops. Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among natural antibodies, H3 and L3 showed the greatest diversity among six HVRs, particularly H3, and are believed to play a unique role in conferring fine specificity to the antibody. See, e.g., Xu et al, Immunity 13: 37-45 (2000); johnson and Wu, Methods in Molecular Biology 248: 1-25(Lo, ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camelid antibodies consisting of only heavy chains are functional, stable, without the presence of light chains. See, e.g., Hamers-Casterman et al, Nature 363: 446-; sheriff et al, Nature struct. biol.3: 733-736(1996).

The HVRs in use are described in a variety of forms, which are included herein. Kabat Complementarity Determining Regions (CDRs) based on sequence variability are most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, Md. (1991)). And Chothia refers to the position of the structural loop (Chothia and Lesk J.mol.biol.196: 901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. The "contact" HVR is based on an analysis of the crystal structure of the available complex. The residues for each of these HVRs are shown below.

The HVRs may include the following "extended HVRs": 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97 or 89-96(L3) in VL, and 26-35(H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al, supra.

"framework" or "FR" residues are those variable domain residues other than the HVR residues as defined herein. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the VH (or VL) in the following order: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof, refers to the numbering system used in Kabat et al above to assemble an antibody heavy chain variable domain or light chain variable domain. Using this numbering system, a substantially linear amino acid sequence may comprise fewer or additional amino acids corresponding to a shortening or insertion of the FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and multiple inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of a given antibody residue can be determined by aligning the regions of homology of the antibody sequence with "standard" Kabat numbered sequences.

When referring to residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al, Sequences of Immunological interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is typically used (e.g., the EU index reported by Kabat et al, supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody.

The expression "linear antibody" refers to the antibody described in Zapata et al (1995Protein Eng,8 (10): 1057-. Briefly, these antibodies comprise a pair of tandemly connected Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions with a complementary light chain polypeptide. Linear antibodies can be bispecific or monospecific.

As used herein, the term "antigen" and grammatical equivalents thereof (e.g., "antigenic") refers to a compound, composition, or substance that can be specifically bound by a particular humoral or cellular immune product (e.g., an antibody molecule or T cell receptor). The antigen may be any type of molecule, including, for example, haptens, simple intermediate metabolites, sugars (e.g., oligosaccharides), lipids, and hormones, as well as macromolecules, such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. Common classes of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoan and other parasitic antigens, tumor antigens, antigens involved in autoimmune diseases, allergies and graft rejections, toxins and other miscellaneous antigens.

As used herein, the terms "bind," "specifically bind," or "specifically" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence or absence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that binds or specifically binds to a target (which may be an epitope) refers to an antibody that binds with greater affinity (affinity) than to other targets, and/or binds more readily than to other targets, and/or binds for a longer duration of time to the target. In one embodiment, the degree of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, e.g., as measured by Radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds a target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In another embodiment, a particular combination may include, but need not necessarily be, an exclusive combination.

As used herein, the term "binding agent" refers to an agent that binds to a target antigen and does not significantly bind to an unrelated compound. Examples of binding reagents that can be effectively used in the disclosed methods include, but are not limited to, lectins, proteins, and antibodies, such as monoclonal, chimeric, or polyclonal antibodies, or antigen-binding fragments thereof, as well as aptamers, Fc domain fusion proteins, and aptamers having or fused to a hydrophobic protein domain (e.g., an Fc domain), and the like. In one embodiment, the binding agent is an exogenous antibody. Foreign antibodies are not antibodies that are naturally produced by the immune system of a mammal, such as a human.

The term "biomarker" as used herein refers to an indicator, such as a predictive, diagnostic and/or prognostic indicator, that can be detected in a sample. Biomarkers can be used as indicators of particular subtypes of diseases or disorders (e.g., T cell dysfunctional disorders) characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number changes (e.g., DNA copy number), polypeptides and polynucleotide modifications (e.g., post-translational modifications), carbohydrate and/or glycolipid based molecular markers.

The terms "biomarker signature," "biomarker expression signature," or "expression signature" are used interchangeably herein and refer to a biomarker or a set of biomarkers whose expression is an indicator (e.g., a predictive, diagnostic, and/or prognostic indicator). Biomarker signatures may be used as indicators of specific subtypes of diseases or disorders (e.g., T cell dysfunctional disorders) characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker signature is a "gene signature". The terms "gene signature" and "gene expression signature" are used interchangeably to refer to a polynucleotide or combination of polynucleotides whose expression is an indicator, e.g., a predictive, diagnostic, and/or prognostic indicator. In some embodiments, the biomarker signature is a "protein signature. The term "protein tag" is used interchangeably with "protein expression tag" and refers to a polypeptide or a group of polypeptides whose expression is an indicator (e.g., a predictive, diagnostic, and/or prognostic indicator).

The terms "cancer" and "cancerous" refer to or describe the physiological condition of a subject that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, anal cancer, penile carcinoma, melanoma, superficial diffuse melanoma, malignant freckle-like melanoma, nodular melanoma, multiple myeloma, and B-cell lymphoma (including low-grade/follicular non-Hodgkin lymphoma (NHL), Small Lymphocytic (SL) NHL, medium/follicular NHL, medium grade NHL, and small cell lymphoma Diffuse NHL; hyperimmune NHL; highly lymphoblastic NHL; highly small non-lytic cellular NHL; mass (bulk disease) NHL; mantle cell lymphoma; AIDS-related lymphomas; and Waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphocytic Leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus (phakomatases), edema (e.g., associated with brain tumors), Meigs syndrome, brain and head and neck cancer, and associated metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. However, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of those cancers. In a particular embodiment, the cancer is melanoma or lung cancer, suitably metastatic melanoma or metastatic lung cancer.

The terms "cell proliferative disorder," "proliferative disorder," and "hyperproliferative disorder" are used interchangeably herein to refer to a disease associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer. In some embodiments, the cell proliferative disorder is a tumor, including a solid tumor.

"chemotherapeutic agents" include compounds useful for the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (b)

Genentech/OSI Pharm.), bortezomib (

Millennium Pharm.), disulfiram, epigallocatechin gallate (epigallocatechin gallate), salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicol (radicicol), lactate dehydrogenase (LDH-A), fosweit (R) (Millennium Pharm.), disulfiram, epigallocatechin gallate (epigallocatechin gallate), and mixtures thereof

AstraZeneca), sunitinib (A), (B), (C), (

pyroxene/Sugen), letrozole (ltr.) (

Novartis), imatinib mesylate (

Novartis), fenamate (Novartis)

Novartis), oxaliplatin (A)

Sanofi), 5-FU (5-fluorouracil), folinic acid, rapamycin (Siro)limus,

Huichi), lapatinib (

GSK572016, Glaxo SmithKline), Lonabub (SCH 66336), Sorafenib (S.E.)

Bayer Labs), gefitinib (b)

AstraZeneca), AG1478, alkylating agents, e.g. thiotepa and

cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodidopa, carboquinone, medopa, and Udopa; ethyleneimine and methyl melamine include hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenin (especially bullatacin and bullatacin); camptothecin (including topotecan and irinotecan); a bromostatin; callystatin; CC-1065 (including its Ardoxazosin, Cazelesin, and Becelesin synthetic analogs); cryptophycins (especially cryptophycins 1 and 8); adrenocortical hormones (including prednisone and prednisolone); cyproterone acetate; 5 a-reductases (including finasteride and dutasteride); vorinostat, romidepsin, pantoprazole, valproic acid, moxystadoxostat (mocetinostat) and; aldesleukin, talcpowder duocarmycin (including the synthetic analogs KW-2189 and CB1-TM 1); eleutherobin (eleutherobin); panduratin; sarcodictyin; sponge chalone; nitrogen mustards, e.g. chlorambucil, chlorophenylpiperazine, chlorophenylphosphoramide, estramustine, ifosfamide, mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan, neonebixin, phenacetin, predostilbene, triphospholAmines (trofosfamide), uracil mustard; nitrosoureas such as carmustine, chlorzotocin, temustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ 1I and calicheamicin ω 1I (Angew chem. intel. ed. Engl. 199433: 183) 186; darnimycin, including danamycin A; bisphosphonates, such as clodronate; epsipramine; and neooncostatin chromophore and related chromophoric protein enediyne antibiotic chromophores), aclacinomycin, actinomycin, aurramycin, azaserine, bleomycin, cactinomycin, karabicin (carabicin), carmomycin (caminomycin), carbamycin, chromomycin (chromomycin), actinoD, daunorubicin, ditorexin, 6-diazo-5-oxo-L-norleucine, norubicin, and/or norubicin,

(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and doxorubicin), epirubicin, isoxabixin, idarubicin, mosaicine, mitomycins such as mitomycin C, mycophenolic acid, nogomycin, olivomycin (olivomycin), pelomycin, porfiromycin, puromycin (puromycin), quelemycin, rodobicin, pronuclidines, streptozotocin, tubercidin, ubenimex, neocarzinostain, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs such as, for example, ancitabine, azacitidine, 6-azapyrimidine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine, doxifradine, enoxitin, floxuridine; androgens such as carpoterone, drotanolone propionate, episterol, mepiquitaxane (mepitiostane), testosterone; anti-adrenalines, such as aminoglutamine, mitotane, trostane; folic acid supplements, such as folic acid; glucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid(ii) a Eniluracil; amsacrine; bisanguin (betransbucil); a bisantrene group; idarubicin ester; desphosphamide (defofamine); dimesine (demecolcine); diazoquinone; etomidazine (elfosmithine); ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate (gallium nitrate); hydroxyurea (hydroxyurea); mushroom polysaccharides (lentinan); lonidamine (lonidainine); maytansinoids, such as maytansine and ansamycin (ansamitocins); mitoguazone; mitoxantrone; (ii) daphnol; nitramines (niterine); pentostatin; phenacetin; pirarubicin; losoxanthraquinone; podophyllinic acid; 2-ethyl hydrazine; (ii) procarbazine;

polysaccharide complexes (jhsnaral Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); tetrahydrofuran (sizofuran); helical germanium (spirogermanium); tenazonic acid (Tenuazonic acid); triazinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecenes (trichothecenes) (particularly T-2 toxin, veracurin A, rhodamine A and snake venom (anguidine)); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; a polycytidysine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J),

(Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and

(docetaxel), docetaxel (doxetaxel); Sanofi-Aventis); chlorambucil;

(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate (MTX)(ii) a Platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;

(vinorelbine); nuntoron (novantrone); (ii) teniposide; edatrexed; daunomycin; aminopterin; capecitabine

Ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); tretinoin, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Chemotherapeutic agents also include (i) anti-hormonal agents that act as modulators or suppressors of the tumor, such as anti-estrogen agents and Selective Estrogen Receptor Modulators (SERMs), including for example tamoxifen (including

Tamoxifen citrate), raloxifene, droloxifene, idoxifene, 4-hydroxyttamoxifen, trioxifene, ketorolifene, LY117018, onapritone (onapristone), and

(toremifene citrate); (ii) aromatase inhibitors which inhibit the enzyme aromatase, which regulates the production of estrogen by the adrenal glands, e.g. 4(5) -imidazole, aminoglutarimide, beta-glucosidase, or beta-glucosidase,

(methyl pregnenolone acetate),

(exemestane); pyroxene), formastin (formastanie), fadrozole (fadrozole),

(vorozole),

(letrozole; Novartis) and

(anastrozole; AstraZeneca), (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, (iii) bunseline, triptorelin, medroxyprogesterone acetate, diethylstilbestrol, pramipexole, flumestosterone, all trans-retinoic acid, fenretinide and troxabine (troxacitabine) (1, 3-dioxolane nucleoside cytosine analogues), (iv) protein kinase inhibitors, (v) lipid kinase inhibitors, (v) antisense oligonucleotides, particularly those which inhibit gene expression in signaling pathways associated with abnormal cell proliferation, such as PKC- α, raf and H-vii), (e.g. Ras expression inhibitors such as VEGF (Ras) ribozymes,

) And inhibitors of HER2 expression; (viii) vaccines, e.g. gene therapy vaccines, e.g.

And

rIL-2; topoisomerase 1 inhibitors, e.g.

And (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.

Chemotherapeutic agents also include antibodies, such as alemtuzumab (Campath), bevacizumab (b

Genentech); cetuximab (

Imclone); panitumumab (A)

Amgen), rituximab (

Genentech/Biogen Idec), pertuzumab (

2C4, Genentech), trastuzumab (

Genentech), tositumomab (Bexxar, Corixia) and antibody drug conjugates, ozomicin gemtuzumab (gemtuzumab ozogamicin, (

Hewlett packard)). Other humanized monoclonal antibodies that are agents with therapeutic potential in combination with the compounds of the invention include: aprezumab (apilizumab), aselizumab (aselizumab), tosituzumab (atlizumab), gemtuzumab (palivizumab), gemtuzumab (bapineuzumab), bivatuzumab (bivatuzumab mertansine), trastuzumab (cantuzumab), ceduzumab (cedilizumab), pegylated certuzumab (certolizumab pegol), cidfutuzumab, cidtuzumab (daclizumab), eculizumab (eculizumab), efolizumab (efalizumab), epratuzumab (epratuzumab), polizumab (erluzumab), fituzumab (feruzumab), aryltumumab (zinzumab), ozuzumab (ozuzumab), ozolouzumab (gemtuzumab ozolomide), zepindolizumab (eppenduzumab), pollizumab (erluzumab), fituzumab (perilizumab), fiuzumab (arenlizumab), tumumab (azib), zelizumab (zelizumab), tamuzumab (zelizumab), zelizumab (zelizumab), zelizumab (zelizumab), tezomuzumab (e (zelizumab), tezomuzumab), tezom, Numavizumab (numavizumab) and ocrelizumab (ocr)elizumab, omalizumab (omalizumab), palivizumab (palivizumab), paclobutrazumab (paclobuzumab), perlizumab (palivizumab), pefillizumab (percluzumab), pelizumab (ranibizumab), resivizumab (resivizumab), resilizumab (resilizumab), resilizumab (resivizumab), rolizumab (rovelizumab), rulipuzumab (ruliplizumab), sibrotuzumab (sibutruzumab), sibutruzumab (solituzumab), tuzumab (sotuzumab), tetuzumab (tuzumab), tetiazelizumab (uralizumab), sibutruzumab (sibutruzumab), sibutruzumab (solituzumab), tuzumab (solituzumab), tetuzumab (tuzumab), tetiazetuzumab (tuzumab), tetuzumab (tuzumab), tetuzumab), tetiazizumab (tuzumab), trastuzumab (tuzumab), trastuzumab (trastuzumab), trastuzumab (tuzumab), trastuzumab (trastuzumab), trastuzumab (trastuzumab), trastuzumab (trastuzumab), and (trastuzumab), vislizumab (visilizumab) and anti-interleukin 12(ABT-874/J695, wyeth research and Abbott Laboratories), a recombinant full-length igg.sub.1.lamda antibody of human-only sequence, genetically modified to recognize the interleukin 12p40 protein.

Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or interact directly with EGFR and prevent or reduce its signaling activity, or are referred to as "EGFR antagonists. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb579(ATCC CRL HB 8506), MAb 455(ATCC CRL HB8507), MAb 225(ATCC CRL 8508), MAb 528(ATCC CRL 8509) (see, U.S. patent No. 4,943,533, Mendelsohn et al) and variants thereof, such as chimeric 225(C225 or cetuximab;

) And remodeled human 225(H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human antibody targeting EGFR (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); and humanized and chimeric antibodies that bind EGFR, e.g.U.S. Pat. No. 5,891,996, human antibodies that bind EGFR, such as ABX-EGF or Panitumumab (Panitumumab, see WO98/50433, Abgenix/Amgen), EMD55900(Stragliotto et al, Eur. J.cancer 32A: 636-640(1996)), EMD7200 (matuzumab), a humanized EGFR antibody against EGFR that competes with EGF and TGF- α for EGFR binding (EMD/Merck), human EGFR antibodies, HuMax-EGFR (GenMab), fully human antibodies referred to as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3, and described in U.S. Pat. No. 6,235,883, MDX-447 (Medrex), and humanized or Johnown (Johnson et al) Pat. No. 5,806, WO 35599,358642, WO 435445, WO 35598,3546,3546,48, WO 25,3546,3546,46,46,46,54, WO 25,46,46,46,46,46,54, WO 25, WO 5,598,597,46,46,46,46,54, WO 25,46,54,54, WO 5,597,46,46,46,46,54,48, WO 5,46,48, WO 5,48, WO 5,597,45,45,44,44,45,45,45,45,45,45,435445,598,45,45,150,150, WO 5,598,435445,435445,150, WO 5,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,150,

Genentech/OSI Pharmaceuticals); PD 183805(CI 1033, 2-propenamide, N- [4- [ (3-chloro-4-fluorophenyl) amino)]-7- [3- (4-morpholinyl) propoxy]-6-quinazolinyl]-, dihydrochloride, feverfew); ZD1839, gefitinib

4- (3 '-chloro-4' -fluoroanilino (luoroanilino)) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, AstraZeneca); ZM 105180 ((6-amino-4- (3-methylphenyl-amino) -quinazoline, Zeneca); BIBX-1382(N8- (3-chloro-4-fluorophenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimidinyl [5, 4-d)]Pyrimidine-2, 8-diamine, Boehringer Ingelheim); PKI-166((R) -4- [4- [ (1-phenylethyl) amino)]-1H-pyrrolo [2,3-d]Pyrimidin-6-yl]-phenol) -; (R) -6- (4-)Hydroxyphenyl) -4- [ (1-phenylethyl) amino]-7H-pyrrolo [2,3-d]Pyrimidines); CL-387785(N- [4- [ (3-bromophenyl) amino)]-6-quinazolinyl]-2-butynylamide); EKB-569(N- [4- [ (3-chloro-4-fluorophenyl) amino group]-3-cyano-7-ethoxy-6-quinolinyl]-4- (-dimethylamino) -2-butenamide) (wheaten); AG1478 (fevered); AG1571(SU 5271; pfeiffer); EGFR/HER2 tyrosine kinase dual inhibitors, such as lapatinib (A: (B))

GSK572016 or N- [ 3-chloro-4- [ (3-fluorophenyl) methoxy]Phenyl radical]-6[5[ [ [ 2-methylsulfonyl) ethyl group]Amino group]Methyl radical]-2-furyl]-4-quinazolinamines).

Chemotherapeutic agents also include "tyrosine kinase inhibitors" which include EGFR-targeting drugs as described in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from martial arts; CP-724, 714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (feverfew and OSI); dual HER inhibitors, such as EKB-569 (available from hewlett-packard), may preferentially bind EGFR, but inhibit both HER2 and EGFR-overexpressing cells; lapatinib (GSK 572016; available from Kulanin Scker), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Nowa corporation); pan-HER inhibitors, such as canertinib (CI-1033; Pharmacia); raf-1 inhibitors, such as the antisense agent ISIS-5132 available from ISIS pharmaceuticals, which inhibit Raf-1 signaling; non-HER targeted TK inhibitors, e.g. imatinib mesylate (

Available from the Puerarin Schker company); multiple target tyrosine kinase inhibitors, e.g. sunitinib (C)

Available from pfeiy corporation); VEGF receptor tyrosine kinase inhibitors, such as vatalanib (PTA787/ZK222584, available from Nowa corporation/Schering AG); CI-1040, an inhibitor of MAPK extracellular regulated kinase I (available from Pharmacia); quinazolines, e.g. PD153035,4- (3-chlorobenzene)Amino) quinazoline; a pyridopyrimidine; a pyrimidopyrimidine; pyrrolopyrimidines such as CGP 59326, CGP 60261, and CGP 62706; pyrazolopyrimidine, 4- (phenylamino) -7H-pyrrolo [2,3-d]A pyrimidine; curcumin (diformylmethane, 4, 5-bis (4-fluoroanilino) phthalimide); tyrphostins containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxaline (U.S. patent No. 5,804,396); trypostins (U.S. patent No. 5,804,396); ZD6474(Astra Zeneca); PTK-787(Novartis/Schering AG); pan-HER inhibitors, such as CI-1033 (pyroxene); affinitac (ISIS 3521; ISIS/Lilly); imatinib mesylate

PKI166 (noval); GW2016(Glaxo SmithKline); CI-1033 (pfeiffer); EKB-569 (Whitman); sematinib (pyrosorib); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11(Imclone), rapamycin (sirolimus,

) (ii) a Or as described in any of the following patent publications: U.S. patent nos. 5,804,396; WO 1999/09016(American Cyanamid); WO1998/43960(American Cyanamid); WO 1997/38983(Warner Lambert); WO1999/06378(Warner Lambert); WO 1999/06396(Warner Lambert); WO 1996/30347 (fevery); WO1996/33978 (Zeneca); WO1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferon, colchicine, metoclopramide, cyclosporine, amphotericin, metronidazole, alemtuzumab, aliskiren formic acid, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, cladribine, clofarabine, dalteptin alpha, dineukin (denileukin), dexrazoxane (dexrazoxane), epoetin alpha, erlotinib, filgrastim, histone acetate, ibritumomab (ibritumomab), interferon alpha 2a, interferon alpha 2b, lenalidomide, levamisole (levamisole), mesna (mesna), methoxsalene, nandrolone (nandolone), nerabine (nellabaine), fertuzumab (nofetumomab), interleukin (opril), velamin), pirimiphos (pirimiphos), pegilate (pegalase), pegalazine (pegavase), pegavase (pegavastatin), pegavastatin (pegavastatin), or (pegavastatin), pegavastatin (e), or (pegavastatin (e, Pirfenitin (pegfilgrastim), pemetrexed disodium (pemetrexed disodium), plicamycin (plicamycin), porphyrin sodium (porfimer sodium), quinacrine (quinacrine), labyrine (rasburicase), sargrastim (sargramostim), temozolomide, VM-26, 6-TG, toremifene, tretinoin (tretinoin), ATRA, valrubicin (valrubicin), zoledronate, and zoledronate and pharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate (tixocortol pivalate), triamcinolone acetonide (triamcinolone acetonide), triamcinolone acetonide (triamcinolone alcohol), mometasone (mometasone), amcinonide (amcinonide), budesonide (budesonide), descinonide (desonide), fluocinonide (flucinonide), fluocinolone acetonide acetate (flucinonide), betamethasone (betamethasone), betamethasone sodium phosphate (betamethasone phosphate), dexamethasone (dexamethosone), dexamethasone sodium phosphate (dexamethosone sodium phosphate), fluocinolone acetonide (fluxolone acetonide), fluxolone (fluxolone, hydrocortisone), hydrocortisone 17-butyrate (hydrocortisone-17-buterol valerate, hydrocortisone 17-valproate, hydrocortisone, hydrasone acetate (fluxolone acetonide), triamcinolone acetonide (E), triamcinolone acetonide (E), triamcinolone acetonide (E-17-D), triamcinolone acetonide (E-17-D), triamcinolone acetonide (E-17-acetate, triamcinolone acetonide), triamcinolone acetonide, triamcinolone acetonBlocking agents for hormone 1(IL-1), e.g., anakinra (Kineret), blocking agents for T-cell co-stimulation, e.g., aberrapu (abatacept) (Orencia), blocking agents for interleukin 6(IL-6), e.g., tollizumab

Interleukin 13(IL-13) blockers, e.g., lebrikizumab, interferon α (IFN) blockers, e.g., rotalizumab (Rontalizumab), β 7 integrin blockers, e.g., rhuMAb β 7, IgE pathway blockers, e.g., anti-Mi prime, secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTal/β 2 blockers, e.g., anti-lymphotoxin α (LTa), radioisotopes (e.g., At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu); various research reagents, such as sulfur platinum (thioplatin), PS-341, phenyl butyrate, ET-18-OCH₃Or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigalloate, theaflavin, flavanol, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors, such as chloroquine; -9-tetrahydrocannabinol (dronabinol,

) β -lapachone, lapachol, colchicine, betulinic acid, acetyl camptothecin, scopoletin and 9-aminocamptothecin, podophyllotoxin, tegafur

Bexarotene

Bisphosphonates, e.g. clodronates (e.g. clodronate)

Or

) Etidronate

NE-58095, zoledronic acid/zoledronic acid salt

Alendronate

Pamidronate salt

Tillofosinate (tirudronate)

Or risedronate (risedronate)

And epidermal growth factor receptor (EGF-R); vaccines, e.g.

A vaccine; perifoscin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS 341); CCI-779; tipifarnib (R11577); olaranib, ABT 510; bcl-2 inhibitors, e.g. orlimeson sodium

Pixantrone (pixantrone); farnesyl transferase inhibitors, e.g. lonafarnib (SCH 6636, SARASAR)^TM) And a pharmaceutically acceptable salt, acid or derivative of any of the above; and combinations of two or more of the above, such as CHOP, are abbreviations for cyclophosphamide, doxorubicin, vincristine, and prednisolone combination therapy; FOLFOX, is oxaliplatin (ELOXATIN)^TM) Abbreviation for treatment regimen combining 5-FU and folinic acid.

Chemotherapeutic agents also include non-steroidal anti-inflammatory drugs having analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of cyclooxygenase enzymes. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin (oxaprozin) and naproxen (naproxen), acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam (piroxicam), meloxicam (meloxicam), tenoxicam (tenoxicam), troxicam (droxicam), lornoxicam (lornoxicam) and isoxicam (isoxicam), fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid and COX-2 inhibitors such as celecoxib, etoricoxib (etoricoxib), lumiracoxib (lumiracoxib), parecoxib (parecoxib), rofecoxib (rofecoxib), felicib (valdecoxib) and valdecoxib (naproxb). NSAIDs are useful for alleviating symptoms of disorders such as rheumatoid arthritis, osteoarthritis, inflammatory joint diseases, ankylosing spondylitis, psoriatic arthritis, reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, post-operative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and renal colic.

As used herein, "concomitant diagnosis" refers to a diagnostic method and/or agent for identifying a subject susceptible to treatment with a particular therapy or monitoring therapy and/or identifying an effective dose of the subject or a subset or other group of subjects. For purposes herein, companion diagnostics refer to reagents, such as reagents for detecting, measuring, or locating a T cell functional biomarker (e.g., as described herein) in a sample. Companion diagnosis refers not only to a reagent but also to one or more tests performed using the reagent.

As used herein, the term "complex" refers to a collection or aggregation of molecules (e.g., peptides, polypeptides, etc.) that are in direct and/or indirect contact with each other. In particular embodiments, "contacting," or more specifically, "direct contact" refers to two or more molecules being in sufficient proximity such that attractive non-covalent interactions (e.g., van der waals forces, hydrogen bonding, ionic and hydrophobic interactions, etc.) dominate the molecular interactions. In such embodiments, a complex of molecules (e.g., peptides and polypeptides) is formed under conditions such that the complex is thermodynamically favorable (e.g., as compared to the non-aggregated or non-complexed state of its constituent molecules). The term "polypeptide complex" or "protein complex" as used herein refers to a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer, dodecamer or higher order oligomer. In a specific embodiment, the polypeptide complex is formed by self-assembly of PKC-theta and ZEB 1.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprising" and "comprises" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or step or group of elements. Thus, use of the terms "comprising," "including," and the like, indicate that the listed elements are required or mandatory, but other elements are optional and may or may not be present. "consisting of … …" is meant to include and be limited to anything following the phrase "consisting. Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that no other elements are present. "consisting essentially of" is meant to include any elements listed after the phrase, as well as being limited to other elements that do not interfere with or affect the activity or effect specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of means that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

The terms "relate" and "relate" generally refer to determining a relationship between one data type and another data type or state. In various embodiments, TBET and/or CXCR3 is expressed or TBET: the EOMES ratio correlates with the presence, absence, or extent of inflammatory or activated states of T cells.

"corresponding to" or "relative" refers to an amino acid sequence that exhibits substantial sequence similarity or identity with respect to a reference amino acid sequence. Typically, an amino acid sequence will exhibit at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to at least a portion of a reference amino acid sequence.

The term "cytolytic activity" as used herein refers to a cell (e.g., CD 8)⁺Cells or NK cells) to lyse the target cells. Such cytolytic activity can be determined using standard techniques, e.g., by radiolabelling the target cell.

The term "cytotoxic agent" as used herein refers to any agent that is harmful to a cell (e.g., causes cell death, inhibits proliferation, or otherwise impedes cell function). Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu); a chemotherapeutic agent; a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Exemplary cytotoxic agents may be selected from the group consisting of antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-a inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In some embodiments, the cytotoxic agent is a taxane. In a representative example of this type, the taxane is paclitaxel or docetaxel. In some embodiments, the cytotoxic agent is a platinum agent. In some embodiments, the cytotoxic agent is an antagonist of EGFR. In a representative example of this type, the antagonist of EGFR is N- (3-ethynylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (e.g., erlotinib). In some embodimentsWherein the cytotoxic agent is a RAF inhibitor. In a non-limiting example of this type, the RAF inhibitor is a BRAF and/or CRAF inhibitor. In other non-limiting examples, the RAF inhibitor is vemurafenib (vemurafenib). In one embodiment, the cytotoxic agent is a PI3K inhibitor.

As used herein, the term "cytotoxic therapy" refers to a therapy that causes cellular damage, including, but not limited to, radiation, chemotherapy, photodynamic therapy, radiofrequency ablation, anti-angiogenic therapy, and combinations thereof. When applied to cells, cytotoxic therapeutic agents may cause DNA damage.

As used herein, "delay of progression of a disease" or "reducing the rate of progression of a disease" refers to delaying, impeding, slowing, arresting, stabilizing and/or delaying the progression of a disease (e.g., a T cell dysfunctional disorder). The delay may be of varying lengths of time, depending on the history of the disease and/or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may actually include prevention, wherein the individual does not develop the disease. For example, the occurrence of advanced cancer, such as metastasis, may be delayed.

The term "detecting" includes any means of detection, including direct and indirect detection.

The term "diagnosis" as used herein refers to the identification or classification of a molecular or pathological state, disease or disorder (e.g., a T cell dysfunctional disorder). For example, "diagnosing" may refer to identifying a particular type of T cell dysfunctional disorder. "diagnosis" may also refer to the classification of a particular subtype of a T cell dysfunctional disorder, for example, according to histopathological criteria or molecular characteristics (e.g., a subtype characterized by expression of one or a set of biomarkers (e.g., a particular gene or protein encoded by the gene)).

The term "aiding diagnosis" as used herein refers to a method of aiding in the clinical determination of the presence or nature of a particular type of symptom or condition of a disease or disorder (e.g., a T cell dysfunctional disorder). For example, a method of aiding diagnosis of a disease or disorder (e.g., a T cell dysfunctional disorder) may comprise measuring certain biomarkers in a biological sample from the individual.

A "disorder" is any condition that would benefit from treatment, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose a subject to the disorder.

In the case of immune dysfunction, the term "dysfunction" refers to a state of decreased immune response to antigen stimulation. The term includes the common elements of fatigue and/or anergy in which antigen recognition may occur but the subsequent immune response is ineffective in controlling infection or tumor growth.

As used herein, the term "dysfunction" also includes being refractory or unresponsive to antigen recognition, particularly an impaired ability to translate antigen recognition into downstream T cell effector functions (e.g., proliferation, cytokine production (e.g., IL-2, IFN- γ, TNF- α, etc.)) and/or target cell killing.

An "effective amount" is at least the minimum amount necessary to achieve measurable improvement or prevention of a particular disease. An effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also an amount where any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, lessening of severity, or delaying the onset of the disease, including biochemical, histological, and/or behavioral symptoms of the disease, complications, and intermediate pathological phenotypes that arise during the course of disease progression. For therapeutic use, beneficial or desired results include clinical results, such as reducing one or more symptoms caused by the disease, improving the quality of life of a person suffering from the disease, reducing the dosage of another drug required to treat the disease, enhancing the effect of another drug, such as by targeting, delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumors, an effective amount of the drug may have the following effects: reducing the number of cancer cells, reducing the size of a tumor, inhibiting (i.e., slowing to some extent or wishing to stop) infiltration of cancer cells into peripheral organs, inhibiting (i.e., slowing to some extent and wishing to stop) tumor metastasis, inhibiting to some extent tumor growth, and/or alleviating to some extent one or more symptoms associated with a cancer or tumor. In the case of infection, an effective amount of the drug may have the following effects: reducing the titer of a pathogen (bacteria, virus, etc.) in the circulation or tissue, reducing the number of cells infected by the pathogen, inhibiting (i.e., slowing to some extent or wishing to stop) infection of the pathogen by the organ, inhibiting (i.e., slowing to some extent and wishing to stop) growth of the pathogen, and/or alleviating to some extent one or more symptoms associated with the infection. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient for prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical context, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Thus, in the case of administration of one or more therapeutic agents, an "effective amount" may be considered, and if combined with one or more other agents, it is considered that a single agent may be administered in an effective amount, perhaps to achieve or achieve the desired result.

"effective response" of a patient to a drug treatment or "response" of a patient to a drug treatment and similar phrases refer to a clinical or therapeutic benefit given to a patient at risk for a disease or condition (e.g., cancer) or to a patient having a disease or condition (e.g., cancer). In one embodiment, such benefits include any one or more of the following: extending survival (including overall survival and progression-free survival); producing an objective response (including a complete response or a partial response); or ameliorating the signs or symptoms of cancer. A patient "not effectively responsive" to treatment refers to a patient that does not have any of prolonged survival (including overall survival and progression-free survival), does not produce an objective response (including a complete response or partial response), or does not ameliorate the signs or symptoms of cancer.

"enhancing T cell function" refers to inducing, causing or stimulating T cells to have sustained or amplified biological function, or to renew or stimulateExamples of enhanced T cell function include any one or more of increased IFN- γ secretion, increased TNF- α secretion, CD8 relative to pre-intervention levels⁺Increased secretion of IL-2 by T cells, increased proliferation, increased antigen response (e.g., viral, pathogen, or tumor clearance). In some embodiments, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this increase is known to those of ordinary skill in the art.

The term "epithelial phenotype" is understood in the art and may be identified by morphological, molecular and/or functional characteristics. For example, epithelial cells typically have a rounded or pebbled appearance, express the epithelial marker E-cadherin, divide rapidly and/or have a lower level of motility, invasiveness and/or anchorage-dependent growth than mesenchymal cells.

As used herein, the term "epithelial to mesenchymal transition" (EMT) refers to the transition from an epithelial to mesenchymal phenotype, which is the normal process of embryonic development. EMT is also a process that makes epithelial cells, which are ion and fluid transporters, mesenchymal cells of matrix remodeling when damaged. In cancer, this transformation often results in altered cell morphology, increased expression and invasiveness of mesenchymal proteins. Criteria defined for EMT in vitro include loss of polarity of epithelial cells, separation into individual cells and subsequent dispersion after cell motility has been achieved (see Vincent-Salomon et al, Breast cancer Res.2003; 5 (2): 101-. Classes of molecules whose expression, distribution and/or function changes during EMT, and reasonably involves, growth factors (e.g., transforming growth factor- β (TGF- β), wnt), transcription factors (e.g., Snail, SMAD, LEF and nuclear β -catenin), intercellular adhesion axis molecules (cadherin, catenin), cytoskeletal modulators (Rho family), and extracellular proteases (matrix metalloproteinases, plasma zymogen activators) (see Thompson et al, Cancer Research 65,5991-5995, jul.15, 2005). In particular embodiments, EMT refers to the process by which epithelial cancer cells exhibit a mesenchymal phenotype, which may be associated with metastasis. These mesenchymal cells may exhibit reduced adhesion, increased motility and invasiveness, and are resistant to immunotherapeutics, chemotherapeutic agents, and/or radiation therapy (e.g., therapies that target rapidly dividing cells).

The term "epitope" refers to the portion of a molecule that is recognized and bound by an antibody in one or more of its antigen binding regions. Epitopes are usually composed of groups on the surface of molecules (e.g., amino acids or sugar side chains) and have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, the epitope can be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all interaction points between a protein and an interacting molecule (e.g., an antibody) occur almost linearly along the primary amino acid sequence of the protein. A "nonlinear epitope" or "conformational epitope" comprises a non-continuous polypeptide (or amino amine) within an antigenic protein to which an antibody specific for such epitope binds. Once the desired epitope on the antigen has been determined, it is possible to generate antibodies against that epitope, for example using the techniques described in the specification. Alternatively, in the discovery process, the production and characterization of antibodies can elucidate information about the desired epitope. Antibodies that bind to the same epitope can then be competitively screened by this information. One way to achieve this goal is to conduct competition and cross-competition studies to find antibodies that compete with each other or cross-compete for binding to a target antigen (e.g., PD-1), e.g., antibodies that compete for binding to the antigen.

The term "exhausted" refers to T cell exhaustion as a state of T cell dysfunction caused by sustained TCR signaling that occurs in many chronic infections and cancers. It differs from anergy in that it is not produced by incomplete or insufficient signaling, but rather is produced by persistent signaling. It is defined as follows: poor effector function, sustained expression of inhibitory receptors, and a different transcriptional state than functional effector or memory T cells. Fatigue prevents optimal infection and tumor control. Fatigue may arise from external negative regulatory pathways (e.g., immune-regulatory cytokines), as well as internal negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

The term "expression" with respect to a gene sequence refers to transcription of a gene to produce an RNA transcript (e.g., mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.) and, if appropriate, translation of the resulting mRNA transcript into protein. Thus, it is clear from the context that expression of a coding sequence results from transcription and translation of the coding sequence. In contrast, expression of a non-coding sequence results from transcription of the non-coding sequence.

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a sample. "expression" generally refers to a process by which information (e.g., encoding genes and/or epigenetics) is converted into structures present and operable in a cell. Thus, "expression" as used herein may refer to transcription into a polynucleotide, translation into a polypeptide, or even modification of a polynucleotide and/or polypeptide (e.g., post-translational modification of a polypeptide). Transcribed polynucleotides, translated polypeptides or fragments of polynucleotides and/or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered as expressed, whether they are derived from transcripts produced or degraded by alternative splicing, or from post-translational processing of polypeptides, e.g., by proteolysis. "expressed genes" include those that are transcribed into a polynucleotide (mRNA) and then translated into a polypeptide, as well as those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer RNA and ribosomal RNA).

By "increased expression," "increased expression level," or "increased level" is meant increased expression or level of a biomarker in an individual or portion of an individual (e.g., a cell, tissue, or organ) relative to a control, e.g., one or more individuals not suffering from a disease or disorder (e.g., a T cell dysfunctional disorder), or a portion thereof (e.g., a cell, tissue, or organ) or an internal control (e.g., a housekeeping biomarker).

By "reduced expression", "reduced expression level" or "reduced level" is meant a reduction or reduction in the expression of a biomarker in an individual or a portion of an individual (e.g., a cell, tissue or organ) relative to a control, e.g., one or more individuals not suffering from a disease or disorder (e.g., a T cell dysfunctional disorder), or a portion thereof (e.g., a cell, tissue or organ) or an internal control (e.g., a housekeeping biomarker). In some embodiments, the reduced expression is little or no expression.

The term "housekeeping biomarker" refers to a biomarker or a group of biomarkers (e.g., polynucleotides and/or polypeptides) that are typically similarly present in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene. "housekeeping gene" refers herein to a gene or set of genes that encode a protein whose activity is essential for maintaining cell function and is typically found similarly in all cell types.

As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits cell growth in vitro or in vivo. In one embodiment, the growth inhibitory agent is a growth inhibitory antibody that prevents or reduces proliferation of cells expressing an antigen to which the antibody binds. In another embodiment, the growth inhibitory agent may be one that significantly reduces the percentage of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a phase other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include vinca (vincristine and vinblastine), taxanes and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 phase extend to arrest S phase, for example DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds, the molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and Andentine optical drugs," Murakami et al (W.B. Saunders, Philadelphia,1995), e.g., page 13. Taxane(s) (aPaclitaxel and docetaxel) are anticancer drugs, both derived from yew. Docetaxel: (

Rhone-Poulenc Rorer) is derived from Taxus baccata and is paclitaxel (Taxol: (Taxus brevifolia)

Semi-synthetic analogs of Bristol-Myers Squibb). Paclitaxel and docetaxel promote microtubule assembly from tubulin dimers and stabilize microtubules by preventing depolymerization, thereby inhibiting mitosis of cells.

In the context of the present invention, the term "immune effector cell" relates to a cell that exerts an effector function during an immune response. For example, such cells secrete cytokines and/or chemokines, kill microorganisms, secrete antibodies, recognize infected or cancerous cells, and optionally eliminate such cells. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, Natural Killer (NK) cells, lymphokine-activated killer (LAK) cells, neutrophils, macrophages, and dendritic cells.

In the context of the present invention, the term "immune effector function" includes any function mediated by a component of the immune system, which for example results in killing of virus infected cells or tumor cells or inhibition of tumor growth, and/or inhibition of tumor development, including inhibition of tumor spread and metastasis. Preferably, in the context of the present invention, the immune effector function is a T cell mediated effector function. In helper T cells (CD 4)⁺T cells), such functions include recognition of antigens or antigenic peptides by T cell receptors (which antigens or antigenic peptides are derived from antigens in the case of MHC class II molecules), cytokine release and/or CD8⁺Activation of lymphocytes (CTLs) and/or B cells; in the case of CTLs, elimination of MHC class I molecule presentation, including recognition of antigens or antigenic peptides derived from the antigens in the case of MHC class I molecules by T cell receptors, e.g. by apoptosis or perforin-mediated cell lysisDelivered cells (i.e., cells characterized by antigen presented by MHC class I molecules), production of cytokines such as IFN-. gamma.and TNF- α, and specific cytolytic killing of target cells expressing the antigen.

The term "immune response" refers to any detectable response of the immune system of a host mammal to a particular substance (e.g., an antigen or immunogen), such as an innate immune response (e.g., activation of the Toll receptor signaling cascade), a cell-mediated immune response (e.g., a response mediated by T cells, such as antigen-specific T cells and non-specific cells of the immune system), and a humoral immune response (e.g., a response mediated by B cells, such as production and secretion of antibodies into plasma, lymph, and/or interstitial fluid).

The term "immunogenic" refers to the ability of a substance to elicit, stimulate or induce an immune response, including enhanced T cells (e.g., CD 8)⁺T cells), or improve, enhance, augment or prolong an existing immune response against a particular antigen, whether used alone or linked to a carrier, in the presence or absence of an adjuvant.

"immunogenic" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and enhancing the immunogenicity of tumors facilitates the elimination of tumor cells via an immune response. Examples of enhancing tumor immunogenicity include treatment with PKC-theta inhibitors and PD-1 binding antagonists.

The term "infection" refers to the invasion of body tissues by disease-causing microorganisms, their proliferation and the response of body tissues to these microorganisms and their toxins produced. "infection" includes, but is not limited to, viral, prion, bacterial, viroid, parasitic, protozoal, and fungal infections. Non-limiting examples of viruses include retroviridae human immunodeficiency viruses, such as HIV-1 (also known as HTLV-III, LAV or HTLV-III/LAV or HIV-III); and other isolates, such as HIV-LP); picornaviridae (e.g., poliovirus, hepatitis a virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); caliciviridae (Calciviridae) (e.g., strains that cause gastroenteritis, including norwalk virus and related viruses); togaviridae (Togaviridae) (e.g., equine encephalitis virus, rubella virus); flaviviridae (Flaviridae) (e.g., dengue virus, encephalitis virus, yellow fever virus); coronaviridae (e.g., coronaviruses); rhabdoviridae (Rhabdoviridae) (e.g., vesicular stomatitis virus (vesicular stomatis viruses), rabies virus); filoviridae (Filoviridae) (e.g., ebola virus); paramyxoviridae (Paramyxoviridae) (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, Metapneumovirus); orthomyxoviridae (Orthomyxoviridae) (e.g., influenza virus); bunyaviridae (Bunyaviridae) (e.g., Hantaan viruses (Hantaan viruses), bunyaviruses (bunya viruses), phleboviruses (phleboviruses), and endo viruses (Nairo viruses)); arenaviridae (hemorrhagic fever virus); reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); bimaviridae; hepadnaviridae (hepatitis b virus); parvoviridae (parvoviruses); papovaviridae (papillomavirus, polyomavirus); adenoviridae (most adenoviruses); herpesviridae (herpes simplex virus (HSV)1 and 2, varicella zoster virus, Cytomegalovirus (CMV), herpes virus); poxviridae (variola virus, VACV, pox virus); and iridoviridae (e.g., african swine fever virus); representative bacteria known to be pathogenic include pathogenic Pasteurella (e.g., Pasteurella multocida), Staphylococcus (e.g., Staphylococcus aureus), Streptococcus (e.g., Streptococcus pyogenes), Streptococcus agalactiae (e.g., Streptococcus agalactiae), Streptococcus (e.g., Streptococcus pyogenes), Streptococcus faecalis (e.g., Streptococcus bovis (Streptococcus bovis), Streptococcus bovis (e.g., Streptococcus bovis), Neisseria anaerobicus (Streptococcus pneumoniae), Streptococcus pneumoniae (e.g., Streptococcus pneumoniae), and astrovirus (e.g., Streptococcus pneumoniae), and non-classified viruses (e.g., spongiform encephalopathy pathogens, hepatitis-type pathogens (believed to be a defective satellite of hepatitis B virus), non-type a, non-type B hepatitis pathogens (i.e., intratransmissible hepatitis B), and astrovirus Neisseria meningitidis (Neisseria meningitidis), escherichia (e.g. enterotoxigenic escherichia coli (ETEC), enteropathogenic escherichia coli (EPEC), enterohemorrhagic escherichia coli (EHEC) and enteroinvasive escherichia coli (EIEC)), Bordetella (Bordetella species), Campylobacter (Campylobacter species), Legionella (e.g. Legionella pneumoniae (Legionella pneumaphila)), pseudomonas, shigella, vibrio, yersinia, salmonella, haemophilus (e.g. haemophilus influenzae), brucella, Francisella species, bacteroides, Clostridium (e.g. Clostridium difficile, Clostridium perfringens (Clostridium perfringens), Clostridium tetanium (Clostridium tetani), mycobacterium tetanii (Clostridium tetanii), mycobacterium (mycobacterium tuberculosis) (e.g. larvatus), mycobacterium avium (m) Mycobacterium gordonae (M.gordonae), Helicobacter pylori (Helicobacter pylori), Borrelia burgdorferi (Borrelia burgdorferi), Listeria monocytogenes (Listeria monocytogenes), Chlamydia trachomatis (Chlamydia trachomatis), Enterococcus sp (Enterococcus species), Bacillus anthracis (Bacillus anthracyclis), Corynebacterium diphtheriae (Corynebacterium diphtheria), Erysipelothrix erythraea (Erysiophilum), Enterobacter aerogenes (Enterobacteriaceae), Klebsiella pneumoniae (Klebsiella pneumoniae), Fusobacterium nucleatum (Fusobacterium nuciferum), Streptomyces monicola (Streptomyces moniliformis), Streptomyces Treponema (Streptomyces Treponema), Actinoligna (Streptomyces Treponema), Treponema (Treponema pallidum), Treponema pallidum (Treponema pallidum), and Lecke), Leptospira sp. Non-limiting pathogenic fungi include cryptococcus neoformans (cryptococcus neoformans), Histoplasma capsulatum (Histoplasma capsulatum), coccidia cocci (coccidioideminis), bacillus dermatitidis (Blastomyces dermatitidis), Candida albicans (Candida albicans), Candida glabrata (Candida glabrata), Aspergillus fumigatus (Aspergillus fumigatus), Aspergillus flavus (Aspergillus flavus), and sporotrichum schenckii (spodotrix schenckii). Illustrative pathogenic protozoa, helminths, plasmodia, such as Plasmodium falciparum (Plasmodium falciparum), Plasmodium malariae (Plasmodium malariae), Plasmodium ovale (Plasmodium ovale), and Plasmodium vivax (Plasmodium vivax); toxoplasma gondii (Toxoplasma gondii); trypanosoma brucei (Trypanosoma brucei), Trypanosoma cruzi (Trypanosoma cruzi); schistosoma japonicum (Schistosoma haematobium), Schistosoma mansoni (Schistosoma mansoni), and Schistosoma japonicum (Schistosoma japonicum); leishmania (Leishmania donovani); giardia intestinalis (Giardia intestinalis); cryptosporidium parvum (Cryptosporidium parvum); and so on.

As used herein, "instructional material" includes publications, records, diagrams, or any other expression medium that can be used to convey the usefulness of the compositions and methods of the present invention. The instructional material of the kit of the invention may be, for example, fixed on a container containing the therapeutic or diagnostic agent of the invention, or shipped together with a container containing the therapeutic or diagnostic agent of the invention.

As used herein, the term "label" refers to a detectable compound or composition. Labels are typically conjugated or fused, directly or indirectly, to an agent (e.g., a polynucleotide probe or antibody) and facilitate detection of the agent conjugated or fused thereto. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition to produce a detectable product.

The term "leukocyte" or "leukocyte" as used herein refers to any immune cell, including monocytes, neutrophils, eosinophils, basophils, and lymphocytes.

The term "lymphocyte" as used herein refers to a cell of the immune system, which is a type of leukocyte. Lymphocytes include, but are not limited to, T cells (cytotoxic and helper T cells), B cells, and natural killer cells (NK cells). As used herein, the term "tumor infiltrating lymphocytes" refers to lymphocytes present in a solid tumor. The term "circulating lymphocytes" as used herein refers to lymphocytes present in the circulation (e.g., present in the blood).

"memory T effector cells" refers to a subset of T cells, including CTL and helper T cells that have previously encountered and responded to the cognate antigen; thus, the term T cell that undergoes antigen is often used. Such T cells can recognize heterologous microorganisms, such as bacteria or viruses, and cancer cells. Memory T effector cells become "experienced" by encountering antigens in previous infections, encountered cancer, or previous vaccinations. Upon the second encounter with the microbe, the memory T effector cells can replicate to produce a faster, stronger immune response than the immune system that responds to the microbe for the first time. This behavior is used in T lymphocyte proliferation assays that can reveal exposure to specific antigens.

The term "mesenchymal phenotype" is understood in the art and may be identified by morphological, molecular and/or functional characteristics. For example, mesenchymal cells typically have an elongated or spindle-shaped appearance, express the mesenchymal markers vimentin, fibronectin and N-cadherin, divide slowly or not and/or have a relatively high level of motility, invasiveness and/or anchorage-independent growth compared to epithelial cells.

As used herein, the term "mesenchymal to epithelial transformation" (MET) is a reversible biological process involving the transformation of mesenchymal cells from motile, multipolar or spindle-shaped to planar arrays of polarized cells (planar rrarrarays), known as epithelial cells. MET is the inverse process of EMT. MET occurs in normal development, cancer metastasis and induced reprogramming of pluripotent stem cells. In particular embodiments, MET refers to the reprogramming of cells that have undergone EMT to restore one or more epithelial characteristics (e.g., as described above). For example, such cells typically exhibit reduced motility and/or invasiveness and/or divide rapidly, and thus may regain sensitivity to immunotherapeutic and/or cytotoxic agents.

The term "multiplex PCR" refers to a single PCR reaction using more than one primer set for nucleic acids obtained from a single source (e.g., an individual) for the purpose of amplifying two or more DNA sequences in a single reaction.

The terms "patient," "subject," "host," or "individual" used interchangeably herein refer to any subject, particularly a vertebrate subject, even more particularly a mammalian subject, in need of treatment or prevention. Suitable vertebrates falling within the scope of the invention include, but are not limited to, any member of the subfamily chordata, including primates (e.g., humans, monkeys and apes, and including monkey species (e.g., rhesus Macaca fascicularis), and/or rhesus Macaca mulatta (Macaca mulatta)) and baboon (Papio ursinus), as well as marmosets (species from the genus elaeis), squirrel monkeys (species from the genus Saimiri), and marmosets (tamarins) (species from the genus tamarisk (Saguinus)), and chimpanzees, e.g., chimpanzees (pandylotes)), rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits), cattle (e.g., bismus), sheep (e.g., goats), goats (e.g., goats), horses (e.g., goats), goats (e.g., mares), goats (e.g., goats), Canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars, and the like), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, and the like), and fish. Preferred subjects are humans in need of eliciting an immune response, including an immune response with enhanced T cell activation. However, it will be understood that the foregoing terms do not imply the presence of symptoms.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation in a form that allows the biological activity of the active ingredient to be effective and that does not contain other components that have unacceptable toxicity to the subject to which the composition or formulation is to be administered. Such formulations are sterile. "pharmaceutically acceptable" excipients (carriers, additives) are those that can be reasonably administered to a mammalian subject to provide an effective dose of the active ingredient used.

The term "PD-1" as used herein refers to any form of PD-1 and variants thereof that retain at least a portion of the activity of PD-1. Unless otherwise indicated, e.g., by specific reference to human PD-1, PD-1 includes the native sequence PD-1 of all mammalian species, e.g., human, canine, feline, equine, and bovine. An exemplary human PD-1 is found at UniProt accession number Q15116.

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (e.g., PD-L1, PD-L2). In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a particular aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with the signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In some embodiments, the PD-1 binding antagonist reduces a negative costimulatory signal mediated by or produced by a cell surface protein expressed on a T cell mediated by PD-1, thereby causing a reduction in dysfunction (e.g., enhancing an effector response to antigen recognition) in a dysfunctional T cell. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a particular aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another particular aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another particular aspect, the PD-1 binding antagonist is CT-011 (pidilizumab). In another particular aspect, the PD-1 binding antagonist is AMP-224.

In the context of the present invention, the term "priming" refers to the induction of a first contact of a T cell (typically a naive T cell) with its specific antigen (e.g. an antigen presented to the T cell by an antigen presenting cell), which results in the differentiation of the T cell into an effector T cell (e.g. a cytotoxic T cell or a T helper cell).

"radiation therapy" refers to the use of direct gamma or beta radiation to induce sufficient damage to cells to limit their ability to function normally or to destroy cells completely. It will be appreciated that there are many ways in the art to determine the dosage and duration of treatment. Typical treatments are single administrations with typical doses ranging from 10 to 200 units per day (Grays).

As used herein, the term "sample" includes any biological sample that can be extracted, untreated, treated, diluted, or concentrated from a subject. Samples may include, but are not limited to, biological fluids such as whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, feces (i.e., stool), tears, sweat, sebum, nipple aspirate, ductal lavage, tumor exudate, synovial fluid, ascites, peritoneal fluid, amniotic fluid, cerebrospinal fluid, lymph fluid, fine needle aspirate, amniotic fluid, any other bodily fluid, cell lysate, cell secretions, inflammatory fluids, semen, and vaginal secretions. Samples may include tissue samples and biopsy samples, tissue homogenates, and the like. Advantageous samples may include samples that comprise any one or more of the biomarkers taught herein in a detectable amount. Suitably, the sample may be readily obtained by a minimally invasive method, thereby allowing the sample to be removed or isolated from the subject. In certain embodiments, the sample comprises blood, particularly peripheral blood, or a fraction or extract thereof. Typically, the sample comprises blood cells, such as mature, immature or developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomic cells, blood cells, eosinophils, megakaryocytes, macrophages, dendritic cells or natural killer cells, or portions (e.g., nucleic acid or protein portions) of such cells. In particular embodiments, the sample comprises leukocytes, including Peripheral Blood Mononuclear Cells (PBMCs).

As used herein, "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level of interest for comparison. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased body part (e.g., tissue or cell) of the same subject or individual. For example, healthy and/or non-diseased cells or tissues adjacent to a diseased cell or tissue (e.g., cells or tissues adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissues and/or cells of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased body part (e.g., tissue or cell) of an individual that is not the subject or the individual. In even another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated body tissue or cells of an individual that is not the subject or the individual.

"tissue sample" or "cell sample" refers to a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue from fresh, frozen and/or preserved organs, tissue samples, biopsies and/or aspirates; blood or any blood component, such as plasma; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or mesenchymal fluid; cells from any stage in the subject's pregnancy or development. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

As used herein, the term "sequence identity" refers to the degree of identity of a sequence based on nucleotides to nucleotides or based on amino acids to amino acids over a comparison window. Thus, calculating "percent sequence identity" by comparing two optimally aligned sequences over a comparison window, determining the number of positions of the same nucleic acid base (e.g., a, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) present in the two sequences, yielding the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 yields the percent sequence identity. For the purposes of the present invention, "sequence identity" is understood to mean the "percentage match" calculated by an appropriate method. For example, sequence identity analysis can be performed using a DNASIS computer program (version 2.5 for Windows; available from Hitachi software engineering, Inc., of south san Francisco, Calif., USA), using standard defaults used in the reference manual followed by the software.

As used herein, "small molecule" refers to a compound having a molecular weight of less than 3 kilodaltons (kDa), and typically less than 1.5 kDa, and more preferably less than about 1 kDa. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic (carbon-containing) or inorganic molecules. As will be appreciated by those skilled in the art, based on the present description, a wide variety of chemical and/or biological mixture libraries, typically fungal, bacterial or algal extracts, can be screened with any of the assays of the present invention to identify compounds that modulate biological activity. An "organic small molecule" is an organic compound (or an organic compound complexed with an inorganic compound (e.g., a metal)) having a molecular weight of less than 3 kilodaltons, less than 1.5 kilodaltons, or even less than about 1 kDa.

The "stringency" of the hybridization reaction can be readily determined by one of ordinary skill in the art, and is generally an empirical calculation based on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of homology desired between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For more details and instructions on the stringency of hybridization reactions, see Ausubel et al, Current protocols in Molecular Biology, Wiley Interscience Publishers (1995).

As defined herein, "stringent conditions" or "high stringency conditions" can be identified by: (1) washing is carried out using low ionic strength and high temperature, for example 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulphate, at 50 ℃; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer, pH 6.5, containing 750mM sodium chloride, 75mM sodium citrate, at 42 ℃; or (3) hybridization overnight at 42 ℃ in a solution using 50% formamide, 5XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, ultrasonic salmon sperm DNA (50. mu.g/mL), 0.1% SDS, and 10% dextran sulfate, washing in 0.2XSSC (sodium chloride/sodium citrate) at 42 ℃ for 10 minutes, followed by high stringency washing with EDTA-containing 0.1XSSC at 55 ℃ for 10 minutes.

By "sustained response" is meant a sustained effect on reducing tumor growth after cessation of treatment. For example, the tumor size may remain the same or smaller than the tumor size at the beginning of the administration phase. In some embodiments, the duration of the sustained response is at least the same as the duration of treatment, at least 1.5 times, 2 times, 2.5 times, or 3 times the length of the duration of treatment.

The term "synergistic" as used herein refers to a therapeutic effect when a PKC-theta inhibitor is administered in combination with a PD-1 binding antagonist (or vice versa) that is greater than the predicted therapeutic effect that would be additive when the PKC-theta inhibitor and the PD-1 binding antagonist are administered alone. The term "synergistically effective amounts" as applied to PKC-theta inhibitors and PD-1 binding antagonists refers to the amount of each component of the composition (typically a pharmaceutical formulation) effective to enhance immune effector function, including any one or more of: increased recognition by T cell receptors of antigens or antigenic peptides derived from MHC class II molecules, increased cytokine release and/or CD8⁺Activation of lymphocytes (CTL) and/or B cells, increased recognition of antigens or antigenic peptides derived from MHC class I molecules by T cell receptors, increased clearance of cells presented by MHC class I molecules, i.e. said cells are characterized by antigen presentation by MHC class I, e.g. by apoptosis or perforin-mediated cell lysis, increased cytokine such as IL-2,The dose-response curves used to determine synergy in the art are described, for example, by Sande et al (see, edited by A. Goodman et al, the optimal amount of synergy can be determined by varying factors such as dose level, dosing schedule, and response, using 95% confidence, and computer generated models can be used that generate various equivalent dose-response curves from the dose-response curves for combinations of inhibitors and PD-1 binding antagonists at various levels of immune function enhancement, e.g., optimal dose-response curves for enhanced immune function.

A "T cell dysfunctional disorder" is a disorder or condition of T cells characterized by reduced responsiveness to antigenic stimulation. In a specific embodiment, the T cell dysfunctional disorder is one particularly associated with increased inappropriate signaling by PD-1. In another embodiment, a T cell dysfunctional disorder is one in which T cells are anergic or have a reduced ability to secrete cytokines, proliferate or undergo cytolytic activity. In a particular aspect, the reduced response results in ineffective control of the pathogen or tumor expressing the immunogen. Examples of T cell dysfunctional disorders characterized by T cell dysfunction include unresolved acute infections, chronic infections, and tumor immunity.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable therapeutic effects include reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. For example, if one or more symptoms associated with a T cell dysfunctional disorder are alleviated or eliminated, the individual is successfully "treated," including, but not limited to, reducing cancer cell proliferation (or destruction of cancer cells), reducing pathogen infection, alleviating symptoms caused by the disease, improving the quality of life of people with the disease, reducing the dose of other drugs required to treat the disease, and/or prolonging the survival of the individual.

As used herein, the expressions "Treg" and "regulatory T cell", formerly known as suppressor T cell, refer to T lymphocytes that maintain immune tolerance. During the course of an immune response, tregs suppress T cell-mediated immunity and suppress autoreactive T cells that escape negative selection within the thymus. Adaptive Treg cells (termed Th3 or Tr 1 cells) are thought to be produced during the immune response. Naturally occurring Treg cells (CD 4)⁺CD25⁺FoxP3⁺Treg cells) are produced in the thymus and are syngeneic with developing T cells (CD11 c)⁺) And plasma cell-like (CD 123)⁺) Dendritic cell-cell interactions are involved, and dendritic cells are activated by the cytokine Thymic Stromal Lymphopoietin (TSLP). FoxP3 present in naturally occurring Treg cells distinguishes them from other T cells.

As used herein, "tumor" refers to the growth and proliferation (whether malignant or benign) of all neoplastic cells, as well as all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "hyperproliferative disorder," and "tumor" are not mutually exclusive herein.

"tumor immunity" refers to the process by which tumors escape immune recognition and elimination. Thus, as a therapeutic concept, when this escape is mitigated, the tumor immunity is "treated" and the tumor is recognized and attacked by the immune system. Examples of tumor identification include tumor binding, tumor shrinkage, and tumor clearance.

As used herein, an underlined or italicized gene name shall mean a gene, other than the protein product of the gene, which is represented by the gene name without any underlining or italicization. For example, "PKC-theta" refers to a PKC-theta gene, and "PKC-theta" refers to a protein product or a product resulting from transcription and translation and/or alternative splicing of a PKC-theta gene.

Each embodiment described herein should be applied mutatis mutandis to each embodiment unless specifically stated otherwise.

2. Drugs for enhancing T cell function

The invention is based in part on the following determinations: exposure of functionally suppressed T cells of the mesenchymal phenotype to PKC-theta inhibitors results in epigenetic reprogramming of T cells, abrogating the suppression of their immune effector functions, including increased expression of biomarkers of T cell activation and effector competence (e.g., IL-2, IFN-gamma, and TNF-alpha), decreased expression of biomarkers of T cell effector suppression and cancer progression (e.g., ZEB1), and decreased expression of biomarkers of T cell exhaustion (e.g., PD-1 and EOMES), and increased expression of the transcription factor TBET, which increases IFN-gamma production in cells of the adaptive and innate immune systems. The inventors have also discovered that PKC-theta inhibitor-mediated epigenetic reprogramming confers increased sensitivity to reactivation of PD-1 binding antagonists on exhausted T cells.

Thus, in accordance with the present invention, compositions and methods are provided that utilize PKC-theta inhibitors (e.g., inhibitors of PKC-theta kinase activity or inhibitors of PKC-theta nuclear translocation/localization) and PD-1 binding antagonists to enhance immune effector function and/or enhance T cells (e.g., CD 8)⁺T cells) including increased T cell activation and increased sensitivity of exhausted T cells to reactivation of PD-1 binding antagonists. Thus, the methods and compositions of the invention are particularly useful for treating T cell dysfunctional disorders, including cancer and infections.

2.1 PKC-theta inhibitors

PKC-theta inhibitors include and encompass reducing PKC-theta accumulation, function (e.g., enzymatic activity, nuclear translocation/localization, etc.), or stability; or any agent that reduces the expression of PKC-theta, such inhibitors including, but not limited to, small and large molecules, such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, polysaccharides, lipopolysaccharides, lipids, or other organic (carbon-containing) or inorganic molecules.

PKC-theta inhibitors include and encompass reducing the accumulation, function or stability of PKC-theta; or any agent that reduces expression of the PKC-theta gene, such inhibitors including, but not limited to, small and large molecules, such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, polysaccharides, lipopolysaccharides, lipids, or other organic (carbon-containing) or inorganic molecules.

In some embodiments, the PKC-theta inhibitor is an antagonist nucleic acid molecule that functions to inhibit transcription or translation of PKC-theta transcripts. Representative transcripts of this type include nucleotide sequences corresponding to any one of the following sequences: (1) human PKC- Θ nucleotide sequences, as shown in GenBank accession numbers XM _005252496, XM _005252497, XM _005252498, and XM _005252499, (2) a nucleotide sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any of the sequences mentioned in (1); (3) a nucleotide sequence that hybridizes under at least low, medium, or high stringency conditions to a sequence mentioned in (1); (4) a nucleotide sequence encoding any one of the following amino acid sequences: for example, the human PKC-theta amino acid sequence as shown in GenPept accession numbers XP _005252553, XP _005252554, XP _005252555, and XP _ 005252556; (5) a nucleotide sequence encoding an amino acid sequence which shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity with any one of the sequences mentioned in (4); and a nucleotide sequence encoding an amino acid sequence sharing at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any of the sequences mentioned in (4).

Illustrative antagonist nucleic acid molecules include antisense molecules, aptamers, ribozymes, and triplex forming molecules, RNAi, and external guide sequences. Nucleic acid molecules can function as effectors, inhibitors, modulators, and stimulators of a particular activity possessed by a target molecule, or functional nucleic acid molecules can have de novo activity independent of any other molecule.

Antagonist nucleic acid molecules can interact with any macromolecule, such as a DNA, RNA, polypeptide, or carbohydrate strand. Thus, the antagonist nucleic acid molecules may interact with PKC-theta mRNA or genomic DNA of PKC-theta, or they may interact with a PKC-theta polypeptide. In general, antagonist nucleic acid molecules are designed to interact with other nucleic acids based on sequence homology between the target molecule and the antagonist nucleic acid molecule. In other cases, the specific recognition between the antagonist nucleic acid molecule and the target molecule is not based on sequence homology between the antagonist nucleic acid molecule and the target molecule, but rather on the formation of a tertiary structure that allows specific recognition to occur.

In some embodiments, antisense RNA or DNA molecules are used to directly block translation of PKC-theta by binding to a target mRNA and prevent protein translation. Antisense molecules are designed to interact with a target nucleic acid molecule through canonical or non-canonical base pairing. The interaction of the antisense molecule with the target molecule can be designed to facilitate destruction of the target molecule, for example, by rnase H mediated RNA-DNA hybrid degradation. Alternatively, antisense molecules can be designed to interrupt processing functions that normally occur on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are many ways to optimize antisense efficiency by finding the region that is most accessible to the target molecule. Non-limiting methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. In specific examples, the antisense molecule is present at less than or equal to 10^-6、10^-8、10^-10Or 10^-12Dissociation constant (K) of_d) Binding the target molecule. In a specific embodiment, antisense oligodeoxyribonucleotides derived from the translation initiation site (e.g.the region between-10 and + 10) are used.

Aptamers are molecules that interact appropriately in a specific way with a target molecule. Aptamers are typically small nucleic acids 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem loops or G-quartets. Aptamers can bind small molecules, such as ATP and theophylline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers may be present at less than 10^-12The Kds of M binds very tightly to the target molecule. Is suitable forThe aptamer has a molecular weight of less than 10^-6、10^-8、10^-10Or 10^-12K of_dBinding the target molecule. Aptamers can bind target molecules with a very high degree of specificity. For example, aptamers have been isolated that differ by more than a factor of 10,000 in binding affinity between a target molecule and another molecule that differs only at a single position of the molecule. Desirably, the aptamer has a K with the target molecule_dK of aptamer to background binding molecule_dAt least 10, 100, 1000, 10,000, or 100,000 times lower. A suitable method for generating aptamers against a target of interest (e.g., PKC- θ) is "Systematic Evolution of Ligands by EXponential Enrichment" (Systematic Evolution of Ligands by ectopic Enrichment, SELEX^TM)。SELEX^TMThe process is described in U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163 (see also WO 91/19813). Briefly, a mixture of nucleic acids is contacted with a target molecule under conditions that favor binding. Unbound nucleic acid is separated from bound nucleic acid and the nucleic acid-target complex is dissociated. The dissociated nucleic acids are then amplified to produce a ligand-enriched nucleic acid mixture, which is subjected to repeated cycles of binding, separating, dissociating, and amplifying as needed to produce high affinity nucleic acid ligands of high specificity for the target molecule.

In other embodiments, an anti-PKC-theta ribozyme is used to catalyze the specific cleavage of PKC-theta RNA. The mechanism of ribozyme action involves sequence-specific hybridization of a ribozyme molecule to a complementary target RNA followed by endonuclease cleavage. There are several different types of ribozymes that catalyze a ribozyme or nucleic acid polymerase type reaction, which are based on the ribozymes that occur in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena ribozymes. There are also ribozymes which do not exist in the natural system, but which are engineered to catalyze specific reactions which start de novo. Representative ribozymes cleave RNA or DNA substrates. In some embodiments, ribozymes that cleave RNA substrates are used. Specific ribozyme cleavage sites in a potential RNA target are first identified by scanning the target molecule for ribozyme cleavage sites, which include the following sequences, GUA, GUU and GUC. Once identified, predicted structural features, such as secondary structure, of short RNA sequences between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated, which may render the oligonucleotide sequence unsuitable. The suitability of a candidate target can also be assessed by detecting accessibility to hybridization with a complementary oligonucleotide using a ribozyme protection assay.

Triple-stranded (triplex) forming functional nucleic acid molecules are molecules that can interact with double-stranded or single-stranded nucleic acids. When a triplex molecule interacts with a target region, a structure called a triplex is formed in which the three DNA strands form a complex that relies on Watson-Crick and Hoogsteen base pairing. Triple-stranded molecules are preferred because they can bind the target region with high affinity and specificity. It is generally desirable for the triplex forming molecule to be less than 10^-6、10^-8、10^-10Or 10^-12K of_dBinding the target molecule.

An External Guide Sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex that is recognized by RNase P, which cleaves the target molecule. EGS can be designed to specifically target selected RNA molecules. RNase P facilitates the processing of the transfer RNA (tRNA) in the cell. Bacterial RNase P can be recruited by using EGS (to mimic the natural tRNA substrate with the target RNA: EGS complex) to cleave almost any RNA sequence. Similarly, EGS/RNAse P-directed RNA cleavage of eukaryotes can be used to cleave desired targets in eukaryotic cells.

In other embodiments, RNA molecules of PKC-theta genes or PKC-theta transcripts that mediate RNA interference (RNAi) may be used to reduce or eliminate gene expression. RNAi refers to the product of interfering with or destroying a target gene by introducing single-stranded or generally double-stranded rna (dsrna) homologous to the transcript of the target gene. RNAi methods, including double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), have been widely documented in many organisms, including mammalian cells and the nematode C.elegans (Fire et al, 1998.Nature 391, 806-. In mammalian cells, RNAi can be triggered by a 21 to 23 nucleotide (nt) duplex of small interfering RNA (siRNA) (Chiu et al, 2002mol. cell.10: 549-SP 561; Elbashir et al, 2001.Nature 411: 494-498), or by micro-RNA (miRNA), functional small hairpin RNA (shRNA) or other dsRNA expressed in vivo using a DNA template and RNA polymerase III promoter (Zeng et al, 2002.mol. cell 9: 1327-SP 1333; Paddison et al, 2002.Genes Dev.16: 948-958; Lee et al, 2002.Nature Biotechnol.20: 500-Sci 505; Paul et al, 2002.Nature 20: 505-SP-508; schl, T.2002. Nature Biotechnol.20: 440-448; Yu et al, 2002. c. Acronol.2002. 2002. Nature.20: 2002-5547-19; Proad.35-35, USA) 608; Probashi et al, 2002.Nature 608-608; USA) RNA 608-608).

In particular embodiments, the dsRNA itself and in particular dsRNA producing constructs corresponding to at least a portion of a PKC- Θ gene are used to reduce or eliminate its expression. Inhibition of RNAi-mediated gene expression may be accomplished using any technique reported in the art, for example, by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing the transfected nucleic acid construct (which has homology to the PKC-theta gene between convergent (convergent) promoters, or as a head-to-head or tail-to-tail replica behind a single promoter). Any similar construct can be used so long as it produces a single RNA with the ability to fold upon itself and produce dsRNA, or so long as it produces two separate RNA transcripts that then anneal to form dsRNA with homology to the target gene.

RNAi does not require absolute homology, and for dsRNA of about 200 base pairs, a lower threshold is described as about 85% homology (Plasterk and keying, 2000, Current Opinion in Genetics and Dev.10: 562-67). Thus, depending on the length of the dsRNA, the nucleic acid encoding the RNAi can differ in the level of homology it comprises to the transcript of the target gene, i.e., where a 100 to 200 base pair dsRNA has at least about 85% homology to the target gene and longer dsRNA (i.e., 300-100 base pair dsRNA) has at least about 75% homology to the target gene. The RNA-encoding construct is suitably at least about 100 nucleotides long, which expresses a single RNA transcript, is designed to anneal to an individually expressed RNA, or expresses a single construct of an individual transcript from a convergent promoter. RNA-encoding constructs, typically at least about 200 nucleotides long, that express a single RNA, are designed to form dsRNA by internal folding.

The promoter used to express the construct forming the dsRNA may be any type of promoter if the resulting dsRNA is specific for a gene product in a cell lineage targeted for disruption. Alternatively, the promoter may be lineage specific in that it is expressed only in cells of a particular developmental lineage. This lineage specific promoter may be advantageous when some homology overlap is observed in genes expressed by non-targeted cell lineages. Promoters may also be induced by external control factors or intracellular environmental factors.

In some embodiments, RNA molecules of about 21 to about 23 nucleotides can be used to mediate RNAi, which can directly cleave the specific mRNA corresponding thereto, e.g., as described in Tuschl et al, u.s.2002/0086356. Such 21 to 23nt RNA molecules may comprise a 3' hydroxyl group, may be single-stranded or double-stranded (as two 21 to 23nt RNAs), wherein the dsRNA molecule may be blunt-ended or comprise an overhanging end (e.g., 5', 3 ').

In some embodiments, the antagonist nucleic acid molecule is an siRNA. The siRNA may be prepared by any suitable method. For example, reference may be made to international publication WO 02/44321, which discloses sirnas capable of sequence-specific degradation of a target mRNA when paired with a 3' overhang base, which is incorporated herein by reference. Sequence-specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNA (siRNA that mimics the production of the enzyme dicer). sirnas can be chemically synthesized, synthesized in vitro, or the result of short double-stranded hairpin-like rna (shrna) that is processed intracellularly into sirnas. Synthetic sirnas are typically designed using algorithms and conventional DNA/RNA synthesizers. Suppliers include Ambion (Austin, Tex.), Chemgenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), GlenResearch (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA may also be administered using a kit (e.g., SILENCER from Ambion)^TMsiRNA construction kit) was synthesized in vitro.

Production of siRNA from vectors is more commonly accomplished by transcription of short hairpin rna (shrna). Kits are available for generating vectors comprising shRNA, e.g., GENESUPPRESSOR by Imgenex^TMBLOCK-IT from construction kit and Invitrogen^TMInducible RNAi plasmids and lentiviral vectors. In addition, methods for formulating and delivering siRNA to a subject are also well known in the art. See, e.g., US 2005/0282188; US 2005/0239731; US 2005/0234232; US 2005/0176018; US 2005/0059817; US 2005/002052; US 2004/0192626; US 2003/0073640; US 2002/0150936; US 2002/0142980 and US 2002/0120129, each of which is incorporated herein by reference.

Exemplary RNAi molecules (e.g., PKC- θ siRNAs and shRNAs) are described in the art (e.g., Ma et al, 2013.BMC biochem.14: 20; and Kim et al, 2013.Immune NetW.13 (2): 55-62), or are commercially available from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA), OriGene Technologies, Inc. (Rockville, Md., USA), Sigma-Aldrich Pty Ltd (Castle Hill, NSW, Australia).

The invention further contemplates peptide or polypeptide based inhibitor compounds. For example, various PKC-theta isozymes and variable region-specific peptides are known, illustrative examples of which include:

(a) θ V1-derived peptides θ V1-1 and θ V1-2 having the amino acid sequences GLSNFDCG (PKC- θ residues 8-15) or YVESENGQMYI [ SEQ ID NO: 1] (PKC-theta residues 36-46), as disclosed, for example, in U.S. patent No. 5,783,405, the entire contents of which are incorporated herein by reference;

(b) a θ V5 derived peptide having the amino acid sequence VKSPFDCS (PKC- θ residues 655-; and

(c) Ψ θ RACK-derived peptides having the amino acid sequence KGDNVDLI, kgendvdli, KGKEVDLI, KGKNVDLI, RGKNVELA, RGENVELA, KGKQVNLI, KGDQVNLI, or KGEQVNLI, as disclosed, for example, in US2010/0311644, the entire contents of which are incorporated herein by reference.

For example, as described above, PKC-theta inhibitory peptides may be modified by being part of a fusion protein. The fusion protein may comprise a transporter protein or peptide having the function of increasing cellular uptake of a peptide inhibitor, which has another desired biological effect, such as a therapeutic effect, or may have both functions. Fusion proteins can be produced by methods known to the skilled artisan. The inhibitory peptide may be bound or conjugated to another peptide in a variety of ways known in the art. For example, the inhibitory peptide may be conjugated to a carrier peptide or other peptide described herein by cross-linking, wherein both peptides of the fusion protein retain their activity. As another example, peptides may be linked or conjugated to each other through an amide bond from the C-terminus of one peptide to the N-terminus of another peptide. The linkage between the inhibitory peptide and another member of the fusion protein may be a non-cleavable peptide bond, or may be cleaved by, for example, an ester or other cleavable bond as known in the art.

In some embodiments, the transporter protein or peptide may be, for example, a sequence derived from the drosophila antennapedia (drosophila antennapedia) homeodomain comprising amino acid sequence CRQIKIWFQNRRMKWKK [ SEQ id no: 2] and can be linked to an inhibitor by N-terminal Cys-Cys linkage cross-linking (e.g., as discussed in Theodore et al, 1995.J. Neurosci.15: 7158-7167; Johnson et al, 1996.Circ. Res 79: 1086). Alternatively, inhibitors may be modified by trans-activated regulatory protein (Tat) -derived transit polypeptides (e.g., amino acids 47-57 of Tat shown in SEQ ID NO: 3: YGRKKRRQRRR from human type 1 immunodeficiency virus, such as Vives et al, 1997, J.biol.chem, 272: 16010-16017, U.S. Pat. No. 5,804,604 and GenBank accession No. AAT48070, or by using polyarginines as described in Mitchell et al, 2000.J.peptide Res.56: 318-325 and Rolhbard et al, 2000.Nature Med.6: 1253-1257). The inhibitor may be modified by other methods known to those skilled in the art to increase the cellular uptake of the inhibitor.

The PKC-theta inhibitory peptide may also be introduced into a cell by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding the PKC-theta inhibitory peptide. The nucleic acid may be in the form of a recombinant expression vector. The sequence encoding the PKC-theta inhibitory peptide may be operably linked to a transcriptional control element, such as a promoter, in the expression vector. Suitable vectors include, for example, recombinant retroviruses, lentiviruses, and adenoviruses; retroviral expression vectors, lentiviral expression vectors, nucleic acid expression vectors, and plasmid expression vectors. In some cases, the expression vector is integrated into the genome of the cell. In other cases, the expression vector is maintained in the cell in an episomal (episomal) state.

Suitable expression vectors include, but are not limited to, viral vectors, e.g., based on vaccinia virus; poliovirus; adenoviruses (see, e.g., Li et al, Invest Opthalmol Vis Sci 35: 25432549,1994; Borras et al, Gene Ther 6: 515524,1999; Li and Davidson, PNAS 92: 77007704,1995; Sakamoto et al, H Gene Ther 5: 10881097,1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated viruses (see, e.g., Ali et al, Hum Gene Ther 9: 8186,1998, Flannery et al, PNAS 94: 69166921,1997; Bennett et al, Invest Opthalmol Vis Sci 38: 28572863,1997; Jomary et al, Gene Ther 4: 683690,1997, Rolling et al, Hum Genether 10: 641648,1999; Ali et al, Hum Mol Genet.5: 591594,1996; Srivastava in WO93/09239, Samulski et al, J.Vir.63: 3822-; SV 40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al, PNAS 94: 1031923,1997; Takahashi et al, J Virol 73: 78127816,1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as rous sarcoma virus, hayworm sarcoma virus, avian leukemia virus (avian leukemia virus), lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and the like.

The invention also contemplates small molecule agents that reduce the functional activity of PKC-theta (e.g., reduce PKC-theta mediated phosphorylation, inhibit the binding of PKC-theta to the promoter of CD44 or uPAR, reduce the binding of PKC-theta (e.g., active PKC-theta) to chromatin, reduce PKC-theta mediated inhibition of guanine exchange factor GIV/Girdin, reduce PKC-theta mediated inhibition of regulatory T cell function, reduce PKC-theta mediated EMT, etc.).

Small molecule agents useful for reducing PKC-theta functional activity in accordance with the present invention include pyridine derivatives that inhibit PKC-theta functional activity, purine compounds that inhibit PKC-theta functional activity, pyrimidine derivatives that inhibit PKC-theta functional activity, aniline compounds that inhibit PKC-theta functional activity, indole derivatives that inhibit PKC-theta functional activity, and the like.

In some embodiments, the small molecule PKC-theta inhibitor is selected from substituted indole derivatives, as described, for example, in U.S. publication No. 2013/0157980 to Cooke et al, the entire contents of which are incorporated herein by reference. Illustrative derivatives of this type include compounds of formula (I);

or a pharmaceutically acceptable salt or hydrate thereof,

in some embodiments of the compounds of formula (I):

x is CH or N;

r is H or PO₃H₂；

R1 is H or C_1-4An alkyl group; r2 is H or C_1-4An alkyl group; r3 is H, C_1-4Alkyl, CN, Hal or OH; r4 and R5 are each independently H or C_1-4An alkyl group; or R4 and R5 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group.

In other embodiments of the compounds according to formula (I):

x is CH;

r is PO₃H₂；

R1 is H;

r2 is H or C_1-4An alkyl group; r3 is H or C_1-4An alkyl group; r4 and R5 are independently from each other H; or R4 and R5 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group.

In other embodiments of the compounds according to formula (I):

x is CH;

r is H;

r1 is H;

r2 is H or C_1-4An alkyl group; r3 is H or C_1-4An alkyl group; r4 and R5 are independently from each other H; or R4 and R5 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group.

In other embodiments of the compounds according to formula (I):

x is N;

r is PO₃H₂；

R1 is H;

r2 is H or C_1-4An alkyl group;

r3 is H; and

r4 and R5 are independently from each other H; or R4 and R5 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group.

In other embodiments of the compounds according to formula (I):

x is N;

r is PO₃H₂；

R1 is H;

r2 is H or C_1-4An alkyl group;

r3 is H; and

r4 and R5 are each independently H or C_1-4An alkyl group.

In some embodiments, substituted indole derivatives that inhibit PKC-theta functional activity include compounds according to formula (II):

or a pharmaceutically acceptable salt thereof.

In other embodiments, substituted indole derivatives that inhibit PKC-theta functional activity include compounds according to formula (III):

or a pharmaceutically acceptable salt or hydrate thereof.

In yet another embodiment, substituted indole derivatives that inhibit PKC-theta functional activity include compounds according to formula (IV):

or a pharmaceutically acceptable salt thereof.

Representative examples of compounds according to formula (I) include: phosphoric acid mono- [3- [3- (3- (4, 7-diaza-spiro [2.5] oct-7-yl) -isoquinolin-1-yl ] -4- (7-methyl-1-H-indol-3-yl) -2, 5-dioxo-2, 5-dihydro-pyrrol-1-ylmethyl ] ester, monohydrate, 3- [3- (4, 7-diaza-spiro [2.5] oct-7-yl) -isoquinolin-1-yl ] -1-hydroxymethyl-4- (-7-methyl-1H-indol-3-yl) -pyrrole-2, 5-dione or a pharmaceutically acceptable salt thereof, and phosphoric acid mono- {3- (1H-indole) Indol-3-yl) -4- [2- (4-methyl-piperazin-1-yl) -quinazolin-4-yl ] -2, 5-dioxo-2, 5-dihydro-pyrrol-1-ylmethyl } ester or a pharmaceutically acceptable salt thereof.

In other embodiments, the small molecule PKC-theta inhibitor is selected from pyrimidinediamine derivatives, as described, for example, in U.S. publication No. 2013/0143875 to Zhao et al, which is incorporated herein by reference in its entirety. Representative derivatives of this type include compounds according to formula (V):

wherein:

R¹selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, -C (O) OR^1a、–S(O)R^1band-S (O)₂R^1c(ii) a Wherein R is^1a、R^1bAnd R^1cEach independently hydrogen, alkyl or phenylalkyl;

R^a、R^b、R^cand R^dIndependently selected from hydrogen and alkyl;

m is an integer of 1 to 5;

p is an integer from 0 to 6;

R²selected from acyloxy, hydroxy, mercapto, acyl, alkyl, alkoxy, substituted alkyl, substituted alkoxy, amino, substituted amino, aminoacyl, acylamino, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO-alkyl, substituted thioalkoxy, amino-acyl, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, -SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl, -SO₂-heteroaryl and trihalomethyl.

X¹、X²And X³Is CR⁵Or X¹、X²And X³One is N and the others are CR⁵；

R⁵Selected from the group consisting of hydrogen, halogen, alkyl, and substituted alkyl;

R³and R⁴Each occurrence is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxy, thioketo, carboxy, carboxyalkyl, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl and-SO₂-a heteroaryl group; or R³And R⁴A 4 to 8 membered ring which together with the carbon atom to which they are attached form a carbocyclic or heterocyclic ring;

n is an integer of 1 to 3;

Z¹、Z²and Z³Selected from the group consisting of CR⁶R^6aN, O and S;

Z⁴and Z⁵Selected from N, C and CR⁶；

R⁶Selected from the group consisting of hydrogen, halogen, alkyl, and substituted alkyl;

R^6aselected from hydrogen, halogen, alkyl and arylSubstituted alkyl, or do not meet the valence requirement; and

the dotted line represents a single or double bond;

or a salt or solvate or stereoisomer thereof.

In some embodiments of compounds according to formula (V), R^a、R^b、R^cAnd R^dRepresents a lower alkyl group. Illustrative examples of such compounds include those wherein R is^a、R^b、R^cAnd R^dThose compounds which are methyl and have the formula (VI):

in other embodiments of the compounds according to formula (V), X¹、X²And X³Each is CH. These compounds have the following formula (VII):

in other embodiments of the compounds according to formula (V), X¹、X²And X³Each is CH; and m is 2. These compounds have the following formula (VIII):

in other embodiments of the compounds according to formula (V), X¹、X²And X³Each is CH; and m is 1. These compounds have the following formula (IX):

in other embodiments of the compounds according to formula (V), X¹、X²And X³Each is CH; n is 2; and a group R³And R⁴Is hydrogen. These compounds have the following formula (X):

in other embodiments of the compounds according to formula (V), X²Is N, X¹And X³Each is CH. These compounds have the following formula (XI):

in other embodiments of the compounds according to formula (V), X³Is N, X¹And X²Each is CH. These compounds have the following formula (XII):

in other embodiments of the compounds according to formula (V), Z⁴Is C, Z⁵Is N. These compounds have the following formula (XIII):

exemplary compounds of formula V include: n2- (4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (2,2,6, 6-tetramethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (1,2,2,6, 6-p-pentamethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (4H-benzo [ b ] pyrrolo [1,2-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (2,2,6, 6-tetra-methylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (4H-benzo [ b ] pyrrolo [1,2-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (1,2,2,6, 6-pentamethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (4, 4-difluoro-4H-benzo [ b ] tetrazole [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (2,2,6, 6-tetramethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (4, 4-difluoro-4H-benzo [ b ] tetrazole [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (1,2,2,6, 6-pentamethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (4, 4-dimethyl-4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (1,2,2,6, 6-pentamethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (4, 4-dimethyl-4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (2,2,6, 6-tetramethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (5, 5-dimethyl-5H-benzo [ e ] tetrazol [1,5-c ] [1,3] oxazin-9-yl) -5-fluoro-N4- (2,2,6, 6-tetramethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (5, 5-dimethyl-5H-benzo [ e ] tetrazol [1,5-c ] [1,3] oxazin-9-yl) -5-fluoro-N4- (1,2,2,6, 6-pentamethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (8, 9-dihydrospiro [ benzo [ b ] tetrazole [1,5-d ] [1,4] oxazine-4, 1' -cyclobutane ] -8-yl) -5-fluoro-N4- (2,2,6, 6-tetramethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (8, 9-dihydrospiro [ benzo [ b ] tetrazole [1,5-d ] [1,4] oxazin-4, 1' -cyclobutane ] -8-yl) -5-fluoro-N4- (1,2,2,6, 6-pentamethylpiperidin-4-yl) pyrimidine-2, 4-diamine; 5-fluoro-N2- (4-methyl-8, 9-dihydro-4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -N4- (2,2,6, 6-tetramethylpiperidin-4-yl) pyrimidine-2, 4-diamine; 5-fluoro-N2- (4-methyl-8, 9-dihydro-4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -N4- (1,2,2,6, 6-pentamethylpiperidin-4-yl) pyrimidine-2, 4-diamine; n2- (4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- ((1,2-, 2,5, 5-pentamethylpyrrolidin-3-yl) methyl) pyrimidine-2, 4-diamine; n2- (4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- ((2,2,5, 5-tetramethylpyrrolidinyl-3-yl) methyl) pyrimidine-2, 4-diamine; n2- (4, 4-dimethyl-4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- - ((1,2,2,5, 5-pentamethylpyrrolidin-3-yl) methyl) pyrimidine-2, 4-diamine; n2- (4, 4-dimethyl-4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- ((2,2,5, 5-tetramethylpyrrolidinyl-3-yl) methyl) pyrimidine-2, 4-diamine-; n2- (4, 4-dimethyl-4H-benzo [ b ] tetrazol [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- (((3S) -2,2, 5-trimethylpyrrolidin-3-yl) methyl) pyrimidine-2, 4-diamine; and N2- (4, 4-dimethyl-4H-benzo [ b ] tetrazole [1,5-d ] [1,4] oxazin-8-yl) -5-fluoro-N4- - (((((3R) -2,2, 5-trimethylpyrrolidin-3-yl) methyl) pyrimidine-2, 4-diamine,

or a salt or solvate or stereoisomer thereof.

Alternative small molecule PKC-theta inhibitor compounds may be selected from aminopyridine compounds, as described, for example, in U.S. publication No. 2013/0137703 to Maltais et al, which is incorporated herein by reference in its entirety. Non-limiting compounds of this type have the formula (XIV):

or a pharmaceutically acceptable salt thereof

Wherein:

R₁is-H, C1-C3 is an aliphatic group, F or Cl. Ring B is a 5 or 6 membered monocyclic heteroaromatic ring. X is-CH-, -S-or-NR₂–。R₂Absent or is-H. Y is-Y1 or-Q1. Y1 is a C1-10 aliphatic group, optionally and independently substituted with one or more F.

Q1 is phenyl or a 5-6 membered monocyclic heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; q1 is optionally and independently substituted with one or more J_aAnd (4) substitution.

D is ring C or-Q-R₃。

Ring C is a 6-8 membered non-aromatic monocyclic ring having 1-2 nitrogen atoms or an 8-12 membered non-aromatic bridged bicyclic ring system having 1-3 heteroatoms selected from nitrogen and oxygen; ring C is optionally and independently substituted with one or more J_bAnd (4) substitution.

Q is-NH-or-O-.

R₃Is substituted by-OH or-NH₂Substituted C1-10 alkyl; wherein R is₃The three to six methylene units in (a) may optionally form a C3-C6 membered cycloalkyl ring; r3 is further independently optionally and independently replaced by one or more J_eAnd (4) substitution.

Each J_aIndependently F or C1-C6 alkyl.

J_bIs C1-C10 alkyl wherein up to three methylene units are optionally substituted as-O-; wherein C1-C10 alkyl is optionally and independently substituted with one or more J_cSubstitution; or J_bIs C3-C6 cycloalkyl or C5-C6 heteroaryl; or J_bIs phenyl, optionally and independently by J_dSubstitution; or two J on the same carbon atom_bTo form ═ O or spiro C3-C6 cycloalkyl.

Each J_cIndependently is F, -OH or C3-C6 cycloalkyl.

Each J_dIndependently F or Cl.

Each J_eIndependently phenyl, a 5-6 membered monocyclic aromatic or non-aromatic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or two J's on the same carbon atom_eForm spiro C3-C6 cycloalkyl.

u is 0 or 1.

In some embodiments, ring B is pyridyl; ring C is selected from piperidinyl, piperazinyl, diazepanyl (diazepanyl), triazo (triazepanyl), azo (azocanyl), diazo (diazocanyl), triazo (triazocanyl), indolyl, indazolyl (indazolyl), or diazabicyclooctyl; ring C is optionally and independently substituted with one or more J_bThe remaining variables are as described above.

Representative compounds according to formula (XIV) include:

pyrazolopyridine compounds are also contemplated by the present invention, as described, for example, in international publication WO2011/094273 and U.S. publication No. 2013/0053395 to Jimenez et al, each of which is incorporated herein by reference in its entirety. Illustrative derivatives of this type include compounds according to formula (XV);

or a pharmaceutically acceptable salt thereof,

wherein:

t is-NH-or absent;

each J_c1And J_c2Independently is-CN, -F, -Cl, -OR, -CH₂OR OR-CF₃；

Each U₁、U₂And U₃Independently is-H, Z or J_bWherein U is₁、U₂And U₃No more than one of which is-H; or U₁、U₂And U₃Are linked together to form C having 0-1 heteroatoms_1-6Cycloalkyl rings, optionally and independently by one or more J_eSubstitution;

z is Y2-Q2;

y2 is absent or is C_1-6Alkyl, optionally and independently substituted with one or more J_dAnd (4) substitution.

Q2 is absent or is C with 0-1 heteroatoms_3-8Cycloalkyl, optionally and independently by one or more J_eSubstitutions wherein Y2 and Q2 cannot be deleted simultaneously;

each J_bIndependently is-F, -OR, -CN, -CF₃、–N(R)₂、–C(O)N(R)₂、C_1-6Alkyl, optionally and independently substituted with one or more J_aSubstitution;

each J_aIndependently is-F, -OR, -N (R)₂or-C (O) N (R)₂；

Each J_dIndependently is-OR, -CN, -C (O) N (R)₂、–N(R)₂Or F;

each J_eIndependently is C_1-6Alkyl, -OR, -N (R)₂、–CF₃Or F; and

each R is-H or C_1-6An alkyl group.

In some embodiments, there is an achiral center, indicated by x, on the carbon.

In representative compounds according to formula (XV): u shape₁Is Z, U₃Is J_b(ii) a And/or U₁And U₂Is Z, U₃Is J_b(ii) a And/or Y₂Is C₁-C₃Alkyl, optionally and independently substituted with one or more J_dSubstitution, Q2 is absent or is C₃-C₆Alkyl, optionally and independently substituted with one or more J_eSubstituted, each J_dIndependently is-OR OR F; and/or J_bis-OH or-NH₂(ii) a And/or each J_c1And J_C2Independently is-CF 3, -CN, -F or-Cl, or J_c1Is F, and J_c2Is CI; or J_c1Is Cl, J_c2Is F.

Non-limiting examples of compounds according to formula (XV) include compounds represented by the following structures:

in a specific embodiment, the pyrazolopyridine compound of formula (XV) is represented by formula (XVI):

this compound was named (R) -2- ((S) -4- (3-chloro-5-fluoro-6- (1H-pyrazolo [3,4-b ] pyridin-3-yl) pyridin-2-yl) piperazin-2-yl) -3-methylbutan-2-ol or compound 27 (also referred to herein as "C27") in Jimenez et al (2013, j.med.chem.561799-180).

In other embodiments, the small molecule PKC-theta inhibitor is selected from pyrazolopyridine compounds, as described, for example, by Boyall et al in U.S. publication No. 2012/0071494, which is incorporated herein by reference in its entirety. Non-limiting compounds of this type are represented by formula (XVa):

or a pharmaceutically acceptable salt thereof,

wherein:

t is 0,1 or 2;

w is 0 or 1;

each J_CIndependently is-CN, -F, -Cl, -OR, -CH₂OR OR-CF₃；

U is Z or J_b；

Z is Y2-Q2;

y2 is absent or is C_1-6Alkyl, optionally and independently substituted with one or more J_dSubstitution;

q2 is absent or is C with 0-1 heteroatoms_3-8Cycloalkyl, optionally and independently by one or more J_eSubstitutions wherein Y2 and Q2 cannot be deleted simultaneously;

each J_bIndependently is-F, -OR, -CN, -CF₃、–N(R)₂、–C(O)N(R)₂、C_1-6Alkyl, optionally and independently substituted with one or more J_aSubstitution;

each J_aIndependently is-F, -OR-N(R)₂or-C (O) N (R)₂；

Each J_dIndependently is-OR, -CN, -C (O) N (R)₂、–N(R)₂Or F;

each J_eIndependently is-OR, -CF₃、–N(R)₂Or F;

t is-CH₂–、-CH(J_b)–、–C(J_b)₂-, -NH-or-N (J)_b) -; and

each R is-H or C_1-6An alkyl group.

In particular embodiments, compounds according to formula XVa are represented by formula XVa1, as disclosed, for example, by Jimenez et al (2013, j.med.chem.561799-180), the entire contents of which are incorporated herein by reference:

wherein:

R¹independently F, Cl or CF₃(ii) a And

R²independently is H, F, Cl, OH, CN or CH₂OH。

In other embodiments, the small molecule PKC-theta inhibitor is selected from tricyclic pyrazolopyridine compounds, as described, for example, in U.S. patent publication No. 2012/0184534 to Brenchley et al, the entire contents of which are incorporated herein by reference. Non-limiting compounds of this type are represented by formula (XVI):

or a pharmaceutically acceptable salt thereof,

wherein:

R₁is-H, halogen, -OR ', -N (R')₂、–C(O)OR'、–C(O)N(R')₂、–NR'C(O)R'、NR'C(O)OR'、–CN、–NO₂Optionally and independently by one or more J_aSubstituted C_1-10Aliphatic radical, or optionally and independentlyIs covered by one or more J_bSubstituted C_3-8Alicyclic.

R₂is-H, halogen, -CN, -NO₂、–OR'、–N(R')₂、–C(O)OR'、–C(O)N(R')₂Optionally and independently substituted by one OR more J, -NR ' C (O) R ', -NR ' C (O) OR_aSubstituted C_1-10Aliphatic radical, or optionally and independently by one or more J_bSubstituted C_3-8Alicyclic.

X is-C-or-N-.

R^xAbsent or is-H.

Ring B is a 5-membered monocyclic heteroaromatic ring, optionally fused to an aromatic or non-aromatic ring; ring B is optionally substituted with one Y and independently further optionally and independently with one or more J_cAnd (4) substitution.

Y is-Y1-Q1.

Y1 is absent or is C_1-10Aliphatic radical in which up to three methylene units of Y1 are optionally and independently substituted by G ', wherein G ' is-O-, -C (O) -, -N (R '), -S (O)_p-; y1 is optionally and independently bound by one or more J_dAnd (4) substitution.

Q1 is absent, or is C having 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur_3-8Monocyclic that is either saturated, partially unsaturated, or fully unsaturated; q1 is optionally and independently substituted with one or more J_bSubstitution; wherein Y1 and Q1 are not simultaneously deleted.

Ring C is a 3-8 membered saturated, partially unsaturated, or fully unsaturated monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-12 membered saturated, partially unsaturated, or fully unsaturated bicyclic ring system having 0-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur; ring C is optionally substituted with one Z and independently further optionally and independently with one or more J_bAnd (4) substitution.

Z is-Y2-Q2.

Y2 is absent or is C_1-10Aliphatic radical in which up to three methylene units of Y2 are optionally and independently substituted by G ', wherein G' is-O-, -C (O) -, -N (R') -, or-S (O)_p-; y2 is optionally and independently bound by one or more J_dAnd (4) substitution.

Q2 deleted, C having 0-3 heteroatoms independently selected from nitrogen, oxygen and sulfur_3-8A monocyclic ring which is saturated, partially unsaturated or fully unsaturated, or an 8-12 membered saturated, partially unsaturated or fully unsaturated bicyclic ring system having 0-5 heteroatoms independently selected from nitrogen, oxygen and sulfur; q2 is optionally and independently substituted with one or more J_eSubstitution; wherein Y2 and Q2 are not simultaneously deleted.

Each R' is independently-H or optionally and independently substituted with one or more J_aSubstituted C_1-6An alkyl group.

Each J_aIndependently halogen, -OR, -N (R)₂、–C(O)OR、–C(O)N(R)₂、–NRC(O)R、–NRC(O)OR、–CN、–NO₂Or oxo.

Each J_bIndependently halogen, -OR, -N (R)₂、–C(O)OR、–C(O)N(R)₂、–NRC(O)R、–NRC(O)OR、–CN、–NO₂Oxo or optionally and independently by J_aSubstituted C1-C6 alkyl.

Each J_cIndependently halogen, -OR ', -N (R')₂、–C(O)OR'、–C(O)N(R')₂、–NR'C(O)R'、–NR'C(O)OR'、–CN、–NO₂Or optionally and independently by one or more J_aA substituted C1-C10 aliphatic radical, or optionally and independently substituted with one or more J_bA substituted C3-C8 cycloaliphatic.

Each J_dIndependently halogen, -CN or-NO₂. Each J_eIndependently of each other halogen, -CN, -NO₂Oxo, C1-10 aliphatic radical, wherein up to three methylene units are optionally and independently substituted with G ', wherein G ' is-O-, -C (O) -, -N (R '), -or-S (O)_pAnd the aliphatic radical is optionally and independently substituted by one or more J_dSubstituted, or J_eIs C_3-8Alicyclic, optionally and independently by one or more J_bAnd (4) substitution.

Each R is independentlyThe root of the earth is-H or C_1-6An alkyl group.

Each p is independently 0,1 or 2.

Representative examples of compounds according to formula (XVI) include:

other embodiments of small molecule PKC-theta inhibitors include 2- (amino substituted) -4-arylpyrimidine compounds, such as described by Fleming et al in U.S. patent publication No. 2011/0071134, the entire contents of which are incorporated herein by reference in their entirety. Representative compounds of this type are represented by formula (XVII):

or a pharmaceutically acceptable salt thereof,

wherein:

R¹and R²Each independently is H, C_1-3Alkyl or C_3-5A cycloalkyl group;

R³is H or F;

R⁴is H, F, -OR^a、–C(O)R^a、–C(O)OR^aor-N (R)^a)；R³And R⁴Together with the carbon atom to which they are attached form a carbonyl group; wherein R is present at each occurrence^aIndependently H, C_1-3Alkyl or C_3-5A cycloalkyl group;

ring A is optionally substituted with 1 or 2 independently occurring R⁵Substituted, wherein each R⁵Independently selected from halogen, C_1-4Aliphatic radical, -CN, -OR^b、–SR^C、–N(R^b)₂、–NR^bC(O)R^b、–NR^bC(O)N(R^b)₂、–NR^bCO₂R^C、–CO₂R^b、–C(O)R^b、–C(O)N(R^b)₂、–OC(O)N(R^b)₂、–S(O)₂R^C、–SO₂N(R^b)₂、–S(O)R^C、–NR^bSO₂N(R^b)₂、–NR^bSO₂R^COR optionally by halogen, -CN, -OR^b、–SR^C、–N(R^b)₂、NR^bC(O)R^b、–NR^bC(O)N(R^b)₂、–NR^bCO₂R^C、–CO₂R^b、–C(O)R^b、–C(O)N(R^b)₂、–OC(O)N(R^b)₂、–S(O)₂R^C、–SO₂N(R^b)₂、–S(O)R^C、–NR^bSO₂N(R^b)₂or-NR^bSO₂R^CSubstituted C_1-4An aliphatic radical, wherein, at each occurrence, R^bIndependently is H or C_1-4An aliphatic group; or two R on the same nitrogen atom^bTogether with the nitrogen atom, form a 5-8 membered aromatic or non-aromatic ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms selected from N, O or S; and each occurrence of R^CIndependently is C_1-4An aliphatic group;

Cy¹selected from: a) a 6-membered aryl or heteroaryl ring substituted with W, which is once present in the meta or para position of the ring; or b) a 5 membered heteroaryl ring substituted with once occurring W;

wherein Cy¹R optionally further represented by 1-3 independent occurrences⁶Substituted, wherein each occurrence of R⁶Independently selected from halogen, C_1-8Aliphatic radical, -CN, -OR^b、–SR^D、–N(R^E)₂、–NR^EC(O)R^b、–NR^EC(O)N(R^E)₂、–NR^ECO₂R^D、–CO₂R^b、–C(O)R^b、–C(O)N(R^E)₂、–OC(O)N(R^E)₂、–S(O)₂R^D、–SO₂N(R^E)₂、–S(O)R^D、–NR^ESO₂N(R^E)₂、–NR^ESO₂R^D、–C(＝NH)–N(R^E)₂OR optionally by halogen, -CN, -OR^b、–SR^D、–N(R^E)₂、–NR^EC(O)R^b、–NR^EC(O)N(R^E)₂、–NR^ECO₂R^D、–CO₂R^b、–C(O)R^b、–C(O)N(R^E)₂、–OC(O)N(R^E)₂、–S(O)₂R^D、–SO₂N(R^E)₂、–S(O)R^D、–NR^ESO₂N(R^E)₂、–NR^ESO₂R^Dor-C (═ NH) -N (R)^E)₂Substituted C_1-8An aliphatic radical, wherein, at each occurrence, R^DIs C_1-6Aliphatic radical, R at each occurrence^EIndependently H, C_1-6Aliphatic radical, -C (═ O) R^b、–C(O)OR^bor-SO₂R^b(ii) a Or two R on the same nitrogen atom^ETogether with the nitrogen atom, form a 5-8 membered aromatic or non-aromatic ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms selected from N, O or S;

w is-R⁸、V–R⁸、L₁-R⁷、V-L₁-R⁷、L₁-V–R⁸Or L₁-V-L₂-R⁷(ii) a Wherein: l is₁And L₂Each independently is optionally substituted C_1-6An alkylene chain; v is-CH₂–、–O–、–S–、–S(O)–、–S(O)₂–、–C(O)–、–CO₂–、–NR^E–NR^EC(O)–、–NR^ECO₂–、–NR^ESO₂–、–C(O)N(R^b)–、–SO₂N(R^b)–、–NR^EC(O)N(R^b) -or-oc (o) -; r⁷Is H, halogen, -OH, -N (R)^F)₂、–CN、–OR^G、–C(O)R^G、–CO₂H、–CO₂R^G、–SR^G、–S(O)R^G、–S(O)₂R^G、–N(R^E)C(O)R^G、–N(R^E)CO₂R^G、–N(R^E)SO₂R^G、–C(O)N(R^F)₂、–SO₂N(R^F)₂、–N(R^E)C(O)N(R^F)₂、–OC(O)R^FOr is optionally selected from C_1-10Aliphatic radical, C_6-10Aryl, 3-14 membered heterocyclyl or 5-14 membered heteroaryl, wherein each occurrence of R^FIndependently H, C_1-6Aliphatic radical, C_6-10Aryl, 3-14 membered heterocyclyl, 5-14 membered heteroaryl, -C (═ O) R^b、–C(O)OR^bor-SO₂R^b(ii) a Or two R on the same nitrogen atom^FTogether with the nitrogen atom, form an optionally substituted 5-8 membered aromatic or non-aromatic ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms selected from N, O or S; at each occurrence, R^GIs C_1-6Aliphatic radical, C_6-10Aryl, 3-14 membered heterocyclyl or 5-14 membered heteroaryl; r⁸Is optionally selected from C_1-10Aliphatic radical, C_6-10A substituent of an aryl group, a 3-14 membered heterocyclic group or a 5-14 membered heteroaryl group;

q is a bond, CH₂Or C (═ O);

Cy²is C_6-10An aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl ring, wherein each ring is optionally substituted with 1-3 independent occurrences of R⁹And one occurrence of R¹⁰The substitution is carried out by the following steps,

wherein each occurrence of R⁹Independently selected from C_1-4Aliphatic radical, -N (R)^b)₂Halogen, NO₂、–CN、–OR^b、–C(O)R^a、–CO₂R^a、–SR^C、–S(O)R^C、–S(O)₂R^C、–OS(O)₂R^C–、N(R^b)C(O)R^a、–N(R^b)CO₂R^a、–N(R^b)SO₂R^a、–C(O)N(R^b)₂、–SO₂N(R^b)₂、–N(R^b)C(O)N(R^b)₂、–OC(O)R^aOr optionally substituted by-N (R)^b)₂Halogen, NO₂、–CN、–OR^b、–C(O)R^a、–CO₂R^a、–SR^C、–S(O)R^C、–OS(O)₂R^C、–S(O)₂R^C、–N(R^b)C(O)R^a、–N(R^b)CO₂R^a、–N(R^b)SO₂R^a、–C(O)N(R^b)₂、–SO₂N(R^b)₂、–N(R^b)C(O)N(R^b)₂or-OC (O) R^aSubstituted C_1-4An aliphatic radical, and

R¹⁰selected from phenyl or a 5-6 membered heterocyclyl or heteroaryl ring.

In certain embodiments, the compound of formula XVII is limited by one or more or all of the following:

1) when Cy is substituted by a group of substituents¹Is phenyl substituted in the meta position by W, then:

a) when W is-OMe, R¹、R²、R³And R⁴Each is hydrogen, and Q is a bond, then when ring A is further substituted with R⁵When substituted, R⁵is-CF₃or-C (O) N (R)^b)₂A group other than; and

b) when W is-OMe, R¹、R²、R³And R⁴Each is hydrogen, and Q is-CH₂When is-then Cy²Is not 1H-benzimidazol-1-yl;

2) when Cy is substituted by a group of substituents¹Is phenyl substituted in the para-position by W, R¹、R²、R³And R⁴When each is hydrogen, then:

a) when Q is a bond, W is not: i) -CONH₂；ii)–CONHR⁸Wherein R is⁸Is a substituent optionally selected from phenyl, -alkylphenyl, alkyl or-alkylheterocycle; iii) -CF₃；iv)–SO₂Me；v)–NH₂；vi)-tBu；vii)–CO₂H when Cy is²Is morpholino; viii) -O (phenyl) when Cy is²When is indole; and ix) ionOMe；

b) When Q is-CH₂-when W is not: i) -CONH₂When Cy is²When is optionally substituted imidazole or benzimidazole; ii) -CONHR⁸Wherein R is⁸Is an optionally substituted group selected from phenyl, -alkylphenyl or-alkylheterocycle; iii) -CF₃；iv)–SO₂Me; v) -OH, wherein Cy²Is a 5-10 membered heterocyclyl ring; vi) tBu when Cy²When it is a 5-to 10-membered heterocyclyl ring; and vii) -OMe; and 3) when Cy is present¹For a 5-membered heteroaryl ring, then:

a) when Cy is substituted by a group of substituents¹Is isoxazole, R¹、R²、R³And R⁴Each is hydrogen, Q is a bond, and W is p-fluorophenyl, then Cy²Is a group other than pyridyl or N-pyrrolidinyl;

b) when Cy is substituted by a group of substituents¹Is triazolyl, R¹、R²、R³And R⁴Each is hydrogen, Q is a bond, and W is- (CH)₂)₂N (cyclopentyl) C (O) CH₂(naphthyl), then Cy²Is a group other than N-piperidinyl;

c) when Cy is substituted by a group of substituents¹Is imidazolyl, R¹、R²、R³And R⁴Each is hydrogen, Q is a bond, and W is meta-CF₃When it is phenyl, then R⁶Is C (O) OCH₂CH₃A group other than; and

d) when Cy is substituted by a group of substituents¹When W is p-fluorophenyl, then R is imidazol-5-yl⁶Is a group other than cyclohexyl.

Non-limiting compounds of this type are represented by the following structure:

in other embodiments, the small molecule PKC-theta inhibitor comprises a pyrimidine derivative, as described, for example, in U.S. publication No. 2005/0124640 to cardoz et al, which is incorporated herein by reference in its entirety. Representative compounds of this type are represented by formula (XVIII):

wherein:

R₁is C_1-8Alkyl radical, C_3-7Cycloalkyl radical, C_3-7cycloalkyl-C_1-8Alkyl, naphthyl, quinolyl, aryl-C_1-8Alkyl or heteroaryl-C_1-8Alkyl radical, wherein at each C_1-8In alkyl, methylene may be optionally substituted by-NHC (O) -or-C (O) NH-, and wherein each C_1-8Alkyl optionally substituted by oxo or one or more C_1-3Alkyl substitution, wherein at C_1-8Two alkyl substituents on the same carbon atom of an alkyl group may optionally be combined to form C_2-5An alkylene bridge, and wherein the aryl group is optionally substituted on adjacent carbon atoms by C_3-6Alkylene bridging group, wherein methylene may be optionally substituted by oxygen, -S-, -S (O) -, -SO₂-or-N (R)₆) -substitution;

or R₁Has the following structure:

wherein x andy is independently 0,1, 2,3 or 4, provided that x + y is 2 to 4, z is 0,1 or 2, and one or two CH's in the ring₂The groups may optionally be substituted by-O-, -S-, -S (O) -, -SO₂-or-N (R)₆) Substitution;

wherein each R₁The groups are optionally substituted with one or more of the following groups: c_1-6Alkyl radical, C_3-6Cycloalkyl, halogen, nitro, hydroxy, C_1-6Alkoxy radical, C_1-6Alkylthio, aryl C_1-6Alkyl, aryloxy, arylthio, aminosulfonyl or optionally substituted by one or two C_1-6Alkyl-substituted amino, wherein each aryl is optionally substituted with: one or more C_1-6Alkyl, halogen, nitro, hydroxy, or optionally substituted by one or two C_1-6Alkyl-substituted amino, and wherein at each C_1-6In alkyl, methylene may be optionally substituted by-NHC (O) -or-C (O) NH-, wherein each C_1-6Alkyl optionally substituted with one or more halogens;

R₂selected from the following groups:

wherein:

n is an integer of 3 to 8;

p is an integer from 1 to 3;

q is an integer of 0 to 3;

R₄and R₅Each independently selected from hydrogen and C_1-6Alkyl, aryl C_1-6Alkyl or amidino, in which each aryl group is optionally substituted by one or more C_1-6Alkyl, halogen, nitro, hydroxy or optionally substituted by one or two C_1-6Alkyl-substituted amino, and wherein each C is_1-6Alkyl is optionally substituted by one or more halogens, wherein amidino is optionally substituted by one to three C_1-6Alkyl substitution;

R₆is hydrogen or C_1-6An alkyl group;

wherein each R₂Radical renOptionally one or more C_1-6Alkyl radical, C_1-6Alkoxy, CN, -OH, -NH₂Or halogen substitution;

R₃is halogen, cyano, nitro, C_1-6Alkyl radical, C_1-6Alkoxycarbonyl or aminocarbonyl, each of which is C_1-6Alkyl optionally substituted with one or more halogens;

or a tautomer, pharmaceutically acceptable salt, solvate, or amino-protected derivative thereof,

in some embodiments of the pyrimidine derivative compounds of formula (XVIII):

R₁is aryl-C_1-4Alkyl or heteroaryl-C_1-4Alkyl radical, wherein at each C_1-4In alkyl, methylene may be optionally substituted by-NHC (O) -or-C (O) NH-, and wherein each C_1-4Alkyl optionally substituted by oxo or one or more C_1-3Alkyl substitution, wherein at C_1-4Two alkyl substituents on the same carbon atom of an alkyl group may optionally combine to form C_2-5And wherein aryl is optionally substituted by C on adjacent carbon atoms_3-6Alkylene bridging group, wherein methylene is optionally substituted by oxygen, sulfur or-N (R)₆) -substitution;

or R₁Has the following structure:

wherein x and y are independently 0,1, 2 or 3, provided that x + y is 2 to 3, and z is 0 or 1;

wherein "heteroaryl" is defined as pyridyl, furyl, thienyl, pyrrolyl, imidazolyl or indolyl;

wherein each R₁The groups are optionally substituted with one or more of the following groups: c_1-6Alkyl, Cl, Br, F, nitro, hydroxy, CF₃、–OCF₃、–OCF₂H、–SCF₃、C_1-4Alkoxy radical, C_1-4Alkylthio, phenyl, benzyl, phenoxy, phenylthioRadicals, aminosulfonyl radicals or optionally substituted by one or two C_1-3An alkyl-substituted amino group;

R₂selected from the following groups:

wherein:

n is an integer from 5 to 7;

p is an integer from 1 to 2;

q is an integer of 1 to 2;

R₄and R₅Each independently selected from hydrogen and C_1-6Alkyl, aryl C_1-6An alkyl or amidino group;

R₆is hydrogen;

R₃br, Cl, F, cyano or nitro;

or a tautomer, pharmaceutically acceptable salt, solvate, or amino-protected derivative thereof;

in other embodiments of the pyrimidine derivative compounds of formula (XVIII):

R₁is phenyl-C_1-4Alkyl or naphthyl C_1-2An alkyl group, a carboxyl group,

wherein each R₁The groups are optionally substituted with one or more of the following groups: methyl, Cl, Br, F, nitro, hydroxy, CF₃、–OCF₃、–SCF₃、C_1-4Alkoxy or C_1-4An alkylthio group;

R₂selected from the following groups:

wherein:

R₄and R₅Each independently selected from hydrogen and C_1-3An alkyl or amidino group;

R₃br, Cl, cyano or nitro;

or a tautomer, pharmaceutically acceptable salt, solvate, or amino-protected derivative thereof;

in other embodiments of the pyrimidine derivatives of formula (XVIII):

R₁is phenyl CH₂-

Wherein the phenyl is optionally substituted with one or more of the following groups: methyl, Cl, Br, F, nitro, hydroxy, CF₃、–OCF₃、–SCF₃、C_1-4Alkoxy or C_1-4An alkylthio group;

R₂selected from the following groups:

R₃is a nitro group;

R₄and R₅Each independently selected from hydrogen, methyl or amidino;

or a tautomer, pharmaceutically acceptable salt, solvate, or amino-protected derivative thereof.

Non-limiting examples of pyrimidine derivative compounds of formula (XVIII) are selected from:

4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -2- [ (2-chlorobenzyl) amino]Pyrimidine-5-carboxylic acid ethyl ester; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]-methyl } -5-nitro-N²- [ (2R) -1,2,3, 4-tetrahydronaphthalen-2-yl]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [ (2S) -1,2,3, 4-tetrahydronaphthalen-2-yl]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [ (1R) -1,2,3, 4-tetrahydronaphthalen-1-yl]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]-methyl } -5-nitro-N²- [ (1S) -1,2,3, 4-tetrahydronaphthalen-1-yl]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (4-chlorophenyl) ethyl group]-5-nitropyrimidine (nitropyrimidine) -2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (2-methylphenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (3-methylphenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (4-methylphenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- - (2-fluorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]-methyl } -N²- [2- (3-fluorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (4-fluorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of²- (2-aminobenzyl) -N⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3, 5-dimethoxybenzyl) -5-nitropyrimidine-2-, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [3, 5-bis (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; {3- [ ({2- [ (2-chlorobenzyl) amino group)]-5-Nitropyrimidin (nitropyridin) -4-yl } amino) methyl]Phenyl } methylamine; 2- ({ [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } methyl) phenol; n is a radical of²- (5-amino-2-chlorobenzyl) -N⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitropyrimidine-2, 4-diamine; 4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -2- [ (2-chlorobenzyl) amino]Pyrimidine-5-carboxamide; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chlorobenzyl) -5-fluoropyrimidine-2, 4-diamine; 3- ({ [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } methyl) -N- [2- (2-methylphenyl) ethyl]A benzamide; (1S,2R) -2- ({ [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } methyl) cyclohexanol; (1R,2R) -2- ({ [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } methyl) cyclohexanol; 4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -2- [ (2-chlorobenzyl) amino]Pyrimidine-5-carboxylic acid methyl ester; 4- { [4- ({ [4- (aminomethyl) cyclohexyl]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } -N- [2- (2-methylphenyl) ethyl group]Butyramide; 5- { [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } -N- [2- (2-methylphenyl) ethyl group]Valeramide; 6- { [4- ({ [4- (aminomethyl) RingHexyl radical]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } -N- [2- (2-methylphenyl) ethyl group]Caproamide; (1R,3R) -3- ({ [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } methyl) -4, 4-dimethylcyclohexanol; n is a radical of⁴- ({ 4-cis- [ (dimethyl-amino) methyl group)]Cyclohexyl } methyl) -N²- (1-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of²- [2- (methylthio) benzyl group]-5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; 5-nitro-N⁴- (piperidin-4-ylmethyl) -N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of²- (1-naphthylmethyl) -5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -N²- (1-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- {4- [ (dimethylamino) methyl group]Benzyl group } -N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- {4- [ (dimethylamino) methyl group]Benzyl } -5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- {4- [ (dimethylamino) methyl group]Benzyl group } -N²- (1-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- [ (1-methylpiperidin-4-yl) methyl]-N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2-, 4-diamine; n is a radical of⁴- [ (1-methylpiperidin-4-yl) methyl]-5-nitro-N²- {2- [ (trifluoromethyl) thio ]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- [ (1-methylpiperidin-4-yl) methyl]-N²- (1-naphthylmethyl) -5-nitropyrimidine (nitropyramidine) -2, 4-diamine; n is a radical of²- (2-chlorobenzyl) -N⁴- [ (1-methylpiperidin-4-yl) methyl]-5-nitropyrimidine-2, 4-diamine; n is a radical of²- (2-methoxybenzyl) -N⁴- [ (1-methylpiperidin-4-yl) methyl]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-methoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [2- (trifluoromethyl) benzyl group]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (-2, 4-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-methoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ 4-fluoro-2- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (pyridin-2-ylmethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) -cyclohexyl]Methyl } -N²- (3-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N- (4-bromobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 4-dimethoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ 2-chloro-5- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 5-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [2- (trifluoromethoxy) benzyl group]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chloro-6-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 3-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]-methyl } -N²- (2-furylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (thiophen-2-ylmethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chlorobenzyl) -5-methylpyrimidine-2, 4-diamine; n is a radical of⁴- (6-aminohexyl))-N²- (2-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n- [4- (aminomethyl) benzyl]-N²- (2-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- (7-aminoheptyl) -N²- (2-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [3- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (1-methyl-1-phenylethyl) -5-nitropyrimidine-2, 4-diamine; 4- (4,4' -bipiperidin-1-yl) -N- (2-chlorobenzyl) -5-nitropyrimidin-2-amine; n is a radical of²- (2-chlorobenzyl) -N⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -5-nitropyrimidine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 5-difluorobenzyl) -5-nitropyrimidine-2, -4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²- [4- (difluoromethoxy) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-ethoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [ (1S) -1-phenylethyl)]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-chloro-2-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (4-pentylbenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-butoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 3-dimethoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) -cyclohexyl]Methyl } -N²- (2, 5-dimethoxybenzyl) -5-nitro-pyrimidine-2, 4-diamine; n is a radical of²- (2-chlorobenzyl) -N⁴- [7- (dimethylamino) heptyl]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (1,1' -biphenyl-2-ylmethyl)) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (-4-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 4-difluorobenzyl) -5-nitropyrimidine-2, -4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²- (3-fluoro-4-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 3-difluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- (2-chlorobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]-methyl } -N²- (2, 6-dimethoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 6-difluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ 2-fluoro-3- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-chloro-2-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (1-phenylpropyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [1- (2-chlorophenyl) -1-methylethyl group]-5-nitropyrimidine-2-, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 3-dihydro-1-benzofuran-5-ylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ (1, 5-dimethyl-1H-pyrrol-2-yl) methyl]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-bromobenzyl) -5-nitropyrimidine (nitropyrinidine) -2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 3-dimethylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 4-dimethylbenzyl) -5-nitropyrimidine-2-, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 5-dimethylbenzyl) -5-nitropyrimidine-2, 4-diamine; 2N⁴- { [4- (aminomethyl) RingHexyl radical]Methyl } -N2- [ 2-fluoro-5- (trifluoromethyl) benzyl [ -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N2- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- {2- [ (trifluoromethyl) thio]-benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- - (6-chloro-2-fluoro-3-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chloro-6-fluoro-3-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²-2-naphthyl-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (1-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N N²- [ 2-fluoro-4- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-chloro-2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (5-chloro-2-) methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-chloro-2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]-methyl } -N²- [ 5-fluoro-2- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (-5-chloro-2-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²- (2, 3-difluoro-4-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (-5-fluoro-2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²-1-naphthyl-5-nitropyrimidine-2, 4-diamine; { 4-trans- [ ({2- [ (2-chlorobenzyl) amino group]-5-Nitropyrimidin-4-yl } amino) methyl]Cyclohexyl } methanol; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- (2, 5-dichlorobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- (2, 4-dichlorobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- (2-bromobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (cyclohexylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- [2- (trifluoromethoxy) benzyl group]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- [2- (trifluoromethyl) benzyl group]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (difluoromethoxy) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl-]Methyl } -N²- [3- (difluoromethoxy) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (-2-chloro-4-fluorobenzyl) -5-nitropyrimidine- -2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) -cyclohexyl]Methyl } -N²- (2-chloro-3, 6-difluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (2,3, 5-trifluorobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (2,3,4, 5-tetrafluorobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [ (1R) -1-phenylethyl)]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²-2, 3-dihydro-1H-indan-2-yl-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ (1S) -2, 3-dihydro-1H-indan-1-yl]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ (1R) -2, 3-dihydro-1H-indan-1-yl]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-chloro-1-naphthyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl)Radical) cyclohexyl]Methyl } -N²- (4-methoxy-2-naphthyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²-quinolin-6-ylpyrimidine-2, 4-diamine; n is a radical of⁴- { [ 4-trans- (aminomethyl) cyclohexyl group]Methyl } - - -N²- (2, 5-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [ 4-trans- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 3-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (2-chlorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (3-chlorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chloro-6-phenoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²-2-naphtylpyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- (1-naphthylmethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (pyridin-3-ylmethyl) pyrimidine-2, 4-diamine; 4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -2- [ (2-chlorobenzyl) amino]Pyrimidine-5-carbonitrile; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [4- (dimethylamino) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [ 4-trans- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-bromobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- (7-aminoheptyl) -N²- (2-bromobenzyl-) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- (7-aminoheptyl) -N²- (2, 5-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n- ({4- [ ({2- [ (2-chlorobenzyl) amino)]-5-Nitropyrimidin-4-yl } amino) methyl]Cyclohexyl } methyl-) guanidine; n is a radical of²- (3-aminobenzyl) -M- { - [4 (aminomethyl) cyclohexyl]Methyl } -5-nitro-pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (2-nitrobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ -2- (2-bromophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl)) Cyclohexyl radical]Methyl } -N²- (2-bromobenzyl) -5-chloropyrimidine-2, 4-diamine; (4- { [ (2- { [2- (1H-indol-3-yl) ethyl]Amino } -5-nitropyrimidino-) 4-yl) amino]Methyl } cyclohexyl) methylammonium chloride; n- ({3- [ ({2- [ (2-chlorobenzyl) -amino)]-5-Nitropyrimidin-4-yl } amino) methyl]Cyclohexyl } methyl) guanidine; 3- ({ [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } methyl) phenol; (4- { [ (2- { [2- (1H-imidazol-4-yl) ethyl]Amino } -5-nitropyrimidin-4-yl) amino]Methyl } cyclohexyl) -methylammonium chloride; n is a radical of²- (2-chlorobenzyl) -M- ({ 4-cis- [ (dimethylamino) methyl)]Cyclohexyl } methyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-chloro-N²- (2-chlorobenzyl) pyrimidine-2, 4-diamine; n is a radical of²- (2-chlorobenzyl) -5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (2-phenylethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (3-phenylpropyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N-²- (4-phenylbutyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl-]Methyl } -5-nitro-N²- (2-phenylpropyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (4-methoxyphenyl) ethyl]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (3-methoxyphenyl) ethyl]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (2-methoxyphenyl) ethyl]-5-nitropyrimidine-2, 4-diamine; 4- [ ({2- [ (2-chlorobenzyl) amino group)]-5-Nitropyrimidin-4-yl } amino) methyl]Piperidine-1-carboximide; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3, 5-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- (5-aminopentyl) -N2- (2-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; 2- (benzylamino) -4- (1,4,6, 7-tetrahydro-imidazo [4, 5-c)]Pyridin-5-yl) -5-trifluoromethyl-pyrimidine; 2- (4-chlorobenzylamino) -4- (1,4,6, 7-tetrahydro-imidazo [4, 5-c)]Pyridin-5-yl) -5-nitro-pyrimidine; 2- (2-chlorobenzylamino) -4- (1,4,6,7-Tetrahydro-imidazole [4,5-c]Pyridin-5-yl) -5-nitro-pyrimidine; 2- (benzylamino) -4- (1,4,6, 7-tetrahydro-imidazo [4, 5-c)]Pyridin-5-yl) -5-nitro-pyrimidine; or N⁴- { [ trans-4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [2- (trifluoromethoxy) benzyl group]Pyrimidine-2, 4-diamine.

In some embodiments, the pyrimidine derivative compound of formula (XVIII) is selected from:

N⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [ (2R) -1,2,3, 4-tetrahydronaphthalen-2-yl]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (4-chlorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (3-methylphenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²- [2- (4-methylphenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (3-fluorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (4-fluorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; (1R,3R) -3- ({ [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } methyl) -4, 4-dimethylcyclohexanol; n is a radical of⁴- ({ 4-cis- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -N²- (1-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of²- [2- (methylthio) benzyl group]-5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; 5-nitro-N⁴- (piperidin-4-ylmethyl) -N²- { -2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of²- (1-naphthylmethyl) -5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl-) -N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -N²- (1-naphthyl-methyl) -5-nitropyrimidine-2, 4-diamine;N⁴- {4- [ (dimethylamino) methyl group]Benzyl group } -N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- {4- [ (dimethylamino) methyl group]Benzyl } -5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- [ (1-methylpiperidin-4-yl) methyl]-N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- [ (1-methylpiperidin-4-yl) methyl]-5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-methoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [2- (trifluoromethyl) benzyl group]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, -4-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ 4-fluoro-2- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-bromobenzyl-) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ 2-chloro-5- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 5-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [2- (trifluoromethoxy) benzyl group]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chloro-6-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²- (2, 3-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [3- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of²- (2-chlorobenzyl) -N⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -5-nitropyrimidines-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 5-difluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (-2-ethoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-chloro-2-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (1,1' -biphenyl-2-ylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 4-difluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (-2, 3-difluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 6-difluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ 2-fluoro-3- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-chloro-2-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-bromobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²- (2, 3-dimethylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- - (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (6-chloro-2-fluoro-3-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chloro-6-fluoro-3-methylbenzyl) -5-nitropyrimidinePyridine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²-2-naphthyl-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (1-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-chloro-2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (5-chloro-2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (3-chloro-2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [ 5-fluoro-2- (trifluoromethyl) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (5-chloro-2-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²- (2, 3-difluoro-4-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (5-fluoro-2-methylbenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- (2, -5-dichlorobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- (2-bromobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (cyclohexylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²- [2- (trifluoromethyl) benzyl group]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (difluoromethoxy) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chloro-4-fluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl-) cyclohexyl]Methyl } -N²- (2-chloro-3, 6-difluorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (2,3, 5-trifluorobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²-2, -3-dihydro-1H-indan-2-yl-5-nitropyrimidinePyridine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-chloro-1-naphthyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (4-methoxy-2-naphthyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²-quinolin-6-ylpyrimidine-2, 4-diamine; n is a radical of⁴- { [ 4-trans- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 3-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (2-chlorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (3-chlorophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-bromo-N²-2-naphtylpyrimidine-2, 4-diamine; 4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -2- [ (2-chlorobenzyl) amino]Pyrimidine-5-carbonitrile; n is a radical of⁴- { [ 4-trans- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-bromobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- (7-aminoheptyl) -N²- (2-bromobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- (7-aminoheptyl) -N²- (2, 5-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n- ({4- [ ({2- [ (2-chlorobenzyl) amino)]-5-Nitropyrimidin-4-yl } amino) methyl]Cyclohexyl } methyl) guanidine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (2-nitrobenzyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (2-bromophenyl) ethyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-bromobenzyl) -5-chloropyrimidine-2, 4-diamine; n- ({3- [ ({2- [ (2-chlorobenzyl) amino group)]-5-Nitropyrimidin-4-yl } amino) methyl]Cyclohexyl } methyl) guanidine 3- ({ [4- ({ [4- (aminomethyl) cyclohexyl)]Methyl } amino) -5-nitropyrimidin-2-yl]Amino } methyl) phenol; n is a radical of²- (2-chlorobenzyl) -N⁴- ({ 4-cis- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of²- (2-chlorobenzyl) -5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro compoundradical-N²- (2-phenylethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- (4-phenylbutyl) pyrimidine-2, 4-diamine; or N⁴- { [ trans-4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [2- (trifluoromethoxy) -benzyl]Pyrimidine-2, 4-diamine.

In other embodiments, the pyrimidine derivative compound of formula (XVIII) is selected from:

N⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -N²- (1-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of²- [2- (methylthio) benzyl group]-5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; 5-nitro-N⁴- (piperidin-4-ylmethyl) -N²- { -2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of²- (1-naphthylmethyl) -5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl-) -N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -N²- (1-naphthyl-methyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- {4- [ (dimethylamino) methyl group]Benzyl group } -N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- {4- [ (dimethylamino) methyl group]Benzyl } -5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- [ (1-methylpiperidin-4-yl) methyl]-N²- [2- (methylthio) benzyl group]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- [ (1-methylpiperidin-4-yl) methyl]-5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-methoxybenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [2- (trifluoromethoxy) benzyl group]Pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]-methyl } -N²- (2, 3-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine；N⁴- { [3- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-chlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of²- (2-chlorobenzyl) -N-⁴- ({4- [ (dimethylamino) methyl group)]Cyclohexyl } methyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-bromobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- [2- (methylthio) benzyl-]-5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- {2- [ (trifluoromethyl) thio]Benzyl } -pyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (1-naphthylmethyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2, 3-dichlorobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of⁴- { [4- (aminomethyl) cyclohexyl group]Methyl } -N²- (2-bromobenzyl) -5-nitropyrimidine-2, 4-diamine; n is a radical of²- (2-chlorobenzyl) -5-nitro-N⁴- (piperidin-4-ylmethyl) pyrimidine-2, 4-diamine; or N⁴- { [ trans-4- (aminomethyl) cyclohexyl group]Methyl } -5-nitro-N²- [2- (trifluoromethoxy) -benzyl]Pyrimidine-2, 4-diamine.

Alternative pyrimidine derivatives of PKC-theta inhibitors include the compounds described by Barbosa et al in U.S. publication No. 2010/0318929, which is incorporated herein by reference in its entirety. These compounds are represented by formula (XIX):

r1 is selected from the following groups:

wherein: p is 1,2 or 3; q is 0 or 1, R₅、R₆Each independently selected from: (A) hydrogen, (B) C_1-6Alkyl, or wherein R₅And R₆Together form a methylene bridge which, together with the nitrogen atom between them, forms a 4-to 6-membered ring, one of whichThe methylene groups are optionally substituted with oxygen or nitrogen atoms, and the ring is optionally and independently substituted with one or more of the following groups: (i) c_1-6Alkyl group, (ii) COR₇Wherein R is₇The method comprises the following steps: (a) c_1-6Alkyl, (b) C_1-6Alkoxy group, (C) C_1-6Alkylcarbonyl, (D) C_1-6Alkylsulfonyl, (E) -CONR₈R₉Wherein R is₈And R₉Each independently selected from: (i) hydrogen, (ii) C_1-6An alkyl group; r₂Selected from the following groups: (F) CF3, (G) cyano, (H) CONH₂(I) Halogen or (J) nitro; r₃Selected from the following groups: (A) hydrogen, (B) C_1-6Alkyl, optionally substituted by halogen, (C) C_1-6Alkoxy, optionally substituted by halogen, (D) halogen, R₄Selected from the following groups: (A) heteroaryl optionally substituted by C_1-6Alkyl substitution; (B) aryl or heteroaryl substituted with one or more of (i) C_1-6Alkyl by hydroxy, oxo or NR₁₀R₁₁Is substituted in which R₁₀And R₁₁Each independently selected from the group consisting of: (a) hydrogen, (b) C_1-6Alkyl optionally substituted by hydroxy or CONH₂Substituted, (C) C_1-6Alkylcarbonyl optionally substituted with one or more halogens, (d) C_1-6Alkylsulfonyl, (e) or wherein R₁₀And R₁₁Constituting a methylene bridge which forms a four-to six-membered ring together with the nitrogen atom between them, (ii) CONR₁₂R₁₃Wherein R is₁₂And R₁₃Each independently selected from hydrogen or C_1-6Alkyl group, (iii) SO₂NR₁₂R₁₃Wherein R is₁₂And R₁₃Each independently selected from hydrogen or C_1-6Alkyl, (C) -NR₁₄R₁₅Wherein R is₁₄And R₁₅Each independently selected from: (i) c_1-6Alkylcarbonyl substituted with amino, (ii) or wherein R₁₄And R₁₅Form methylene bridges, form, together with the nitrogen atom between them, four-to seven-membered rings in which one methylene group is replaced by C_1-6Alkyl substituted, and wherein each C_1-6Alkyl being optionally substituted by hydroxy or NR₁₀R₁₁Is substituted in which R₁₀And R₁₁As previously defined, (D) -CONR₁₆R₁₇Wherein R is₁₆And R₁₇Each independently selected from: (i) c_1-6Alkyl radicals, substituted by hydroxy or NR₁₈R₁₉Is substituted in which R₁₈And R₁₉Each independently selected from hydrogen or C_1-6Alkyl, or wherein R₁₈And R₁₉Form a methylene bridge which, together with the nitrogen atom between them, forms a four-to six-membered ring, wherein one methylene group is optionally substituted by oxygen; (E) c₆Alkynyl optionally substituted by amino, C_1-3Alkylamino or di- (C)_1-3Alkyl) amino substitution; a is independently selected from carbon or nitrogen; or a tautomer, pharmaceutically acceptable salt, solvate, or amino-protected derivative thereof.

In this type of illustrative example: r₁Selected from the following groups:

wherein: q is 0 or 1, R₅、R₆Each independently selected from: (A) hydrogen, (B) or wherein R₅And R₆Together form a methylene bridge which, together with the nitrogen atom between them, forms a five to six membered ring, wherein one methylene group is optionally substituted with one nitrogen atom, and the ring is optionally and independently substituted with one or more of the following groups: (iv) c_1-6Alkyl group, (v) COR₇Wherein R is₇Is C_1-6Alkoxy group, (C) C_1-6Alkylcarbonyl, (D) C_1-6An alkylsulfonyl group; r₂Selected from the following groups: (A) cyano, or (B) nitro; r₃Selected from the following groups: (A) c_1-3Alkyl, (B) C_1-3Alkoxy optionally substituted with fluorine, (C) halogen; r₄Selected from the following groups: (A) aryl substituted with one or more of the following groups: (i) c_1-3Alkyl radicals, substituted by hydroxy or NR₂₀R₂₁Is substituted in which R₂₀And R₂₁Each independently selected from the following groups: (f) hydrogen, (g) C_1-3Alkyl, optionally substituted with hydroxyRadical or CONH₂Substituted, (h) or wherein R₂₀And R₂₁Constituting a methylene bridge which forms, together with the nitrogen atom between them, a five-to six-membered ring, (ii) CONH₂，(iii)SO₂NH₂(B) 3-pyridyl optionally substituted by C_1-3Alkyl-substituted, wherein each alkyl is optionally substituted by amino, (C) -NR₂₂R₂₃Wherein R is₂₂And R₂₃Form methylene bridges which, together with the nitrogen atom between them, form a five-to six-membered ring in which one methylene group is replaced by C_1-3Alkyl substitution, wherein each C_1-3Alkyl is optionally substituted by OH or NR₂₀R₂₁Is substituted in which R₂₀And R₂₁As previously defined, (D) -CONR₂₄R₂₅Wherein R is₂₄And R₂₅Each independently selected from: (i) c_1-3Alkyl radical, which is substituted by C_1-3Alkyl amino substitution; a is independently selected from carbon or nitrogen; or a tautomer, pharmaceutically acceptable salt, solvate, or amino-protected derivative thereof.

In other illustrative examples, the compound of formula (XIX) is represented by formula (XIXa)

Wherein:

R₁selected from the following groups:

wherein: q is 0 or 1, R₅、R₆Each independently selected from: (A) hydrogen, (B) C_1-6Alkylcarbonyl, (C) C_1-6An alkylsulfonyl group; r₂Selected from the following groups: (A) cyano, or (B) nitro; r₃Selected from the following groups: (A) CH (CH)₃，(B)OCF₃，(C)Cl；R₄Selected from the following groups:

wherein: r₂₆Selected from the following groups: (A) c_1-3Alkyl radicals, substituted by hydroxy or NR₂₇R₂₈Is substituted in which R₂₇And R₂₈Each independently selected from the group consisting of: (i) hydrogen, (ii) C_1-3Alkyl optionally substituted by hydroxy or CONH₂Substituted, (B) CONH₂，(C)SO₂NH₂(ii) a A is carbon or nitrogen; or a tautomer, pharmaceutically acceptable salt, solvate, or amino-protected derivative thereof.

Also contemplated as small molecule PKC-theta inhibitors are aniline compounds, as described, for example, in U.S. publication No. 2010/0120869 to Ajioka et al, which is incorporated herein by reference in its entirety. Representative compounds of this type are represented by formula (XX):

wherein X in formula XX is aryl or heteroaryl, each of which is substituted by 1-5R¹And (4) substituting the group. Y in formula XX is-O-, -S (O)_n–、–N(R⁴) -and-C (R)⁴)₂-, where the subscript n is 0 to 2. And Z in formula XX is-N or-CH. Each R in the formula XX¹Independently selected from H, halogen, C_1-8Alkyl radical, C_1-6Heteroalkyl group, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkoxy, -OR^1a、–C(O)R^1a、–C(O)OR^1a、–C(O)NR^1aR^1b、–NR^1aR^1b、–SR^1a、–N(R^1a)C(O)R^1b、–N(R^1a)C(O)OR^1b、–N(R^1a)C(O)NR^1aR^1b、–OP(O)(OR^1a)₂、–S(O)₂OR^1a、–S(O)₂NR^1aR^1b、–S(O)₂–C_1-6Haloalkyl, -CN, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. R of formula XX^1aAnd R^1bEach independently is H or C_1-6An alkyl group. Each R of formula XX²Independently of one another H, halogen, C_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, -NR^1aR^1b、–NR^1aC(O)–C_1-6Alkyl, -NR^1aC(O)–C_1-6Haloalkyl, -NR^1a–(CH₂)–NR^1aR^1b、–NR^1a–C(O)–NR^1aR^1bor-NR^1a–C(O)OR^1aOr, adjacent R¹Radical and adjacent R²The groups may combine to form a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group. R of formula XX³is-NR^3aR^3bor-NCO. R of formula XX^3aAnd R^3bEach independently is H, C_1-6Alkyl, -C (O) -C_1-6Alkyl, -C (O) -C_1-6Haloalkyl, - (CH)₂)–NR^1aR^1b、–C(O)–NR^1aR^1b、–C(O)OR^1aC (S) CN, an amino acid residue, a peptide or an oligopeptide. Each R of formula XX⁴Independently is H or C_1-6Alkyl, or when more than one R is present⁴When the radicals are bound to the same atom, R⁴The radicals optionally combining to form C_5-8A cycloalkyl group. The compounds of formula XX also include salts, hydrates and prodrugs thereof.

In some embodiments, the aniline compound of formula XX is represented by formula XXa:

wherein each R of formula XXa^lIndependently of one another H, halogen, C_1-8Alkyl radical, C_1-6Heteroalkyl group, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkoxy, -OR^1a-CN, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, R of formula XXa^3aAnd R^3bEach independently is H, -C (O) -C_1-6Alkyl, amino acid residues, peptides or oligopeptides.

In other embodimentsIn scheme (XXa) each R of formula¹Independently of one another H, halogen, C_1-8Alkyl radical, C_1-6Haloalkyl, C_1-6Haloalkoxy, -C (O) OR^1aCycloalkyl or heteroaryl. Furthermore, each R of the formula XXa²Independently is H, halogen or-NR^1aC(O)–C_1-6An alkyl group. In other embodiments, each R of formula XXa¹Independently H, methyl, n-propyl, isopropyl, tert-butyl, tert-amyl, Cl, Br, CF₃、OCF₃Cyclopentyl, pyrrolyl or CO₂H, and each R²Independently H or Cl. In other embodiments, R of formula XX^3aIs an amino acid residue, and R^3bIs H. Suitably, the amino acid residue is an arginine residue.

In other embodiments, the aniline compound of formula XX is of formula XXb:

in some other embodiments, Y of formula XXb is S. In still other embodiments, Y of formula XXb is O. In some embodiments, each R of formula XXb¹Independently H, methyl, n-propyl, isopropyl, tert-butyl, tert-amyl, Cl, Br, CF₃、OCF₃Cyclopentyl, pyrrolyl or CO₂H. In other embodiments, each R of formula XXb¹Independently is C_1-8Alkyl or cycloalkyl. In other embodiments, each R of formula XXb¹Independently 4-tert-butyl, 4-cyclopentyl or 4-tert-pentyl.

In other embodiments, the small molecule PKC-theta inhibitor is selected from roterin (rottlerin) (also known as malatoxin or 1- [6- [ (3-acetyl-2, 4, 6-trihydroxy-5-methylphenyl) methyl ] -5, 7-dihydroxy-2, 2-dimethyl-2H-1-benzopyran-8-yl ] -3-phenyl-2-propan-1-one, available from Calbiochem, san diego, california, or a derivative or analog thereof, having formula (XXI).

In other embodiments, the small molecule PKC-theta inhibitor comprises a substituted diaminopyrimidine, as disclosed, for example, in U.S. patent application publication US 2005/0222186a1 to Baudler et al, which is incorporated herein by reference in its entirety. These compounds are represented by formula (XXII):

wherein R is¹、R²And R³Independently selected from the group consisting of substituted or unsubstituted phenyl, naphthyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2, 3-triazolyl, indolyl, benzimidazolyl, furyl (furyl), benzofuryl (benzofuryl), thienyl (thienyl), benzothienyl (benzothiazolyl), thiazolyl, isoxazolyl, pyridyl, pyrimidinyl, quinolinyl, and isoquinolinyl; r⁴Is hydrogen or methyl; r⁵Is hydrogen or methyl; a. the¹Is C_1-3Alkylene or ethyleneoxy (-CH)₂–CH₂–O–)；A²Is C_1-3Alkylene or ethyleneoxy (-CH)₂–CH₂-O-); and hydrates, solvates, salts or esters thereof.

Non-limiting examples of such compounds include: [ 1-benzyl (4-piperidinyl) ] {2- [ (2-picolyl) amino ] -5- (3-thienyl) pyrimidin-4-yl } amine; {5- (4-methoxyphenyl) -2- [ (4-pyridylmethyl) amino) pyrimidin-4-yl } [ 1-benzyl- (-4-piperidinyl) ] amine; { 5-phenyl-2- [ (4-picolyl) amino) pyrimidin-4-yl } [ 1-benzyl (4-piperidinyl-1-) ] amine; {5- (4-chlorophenyl) -2- [ (4-picolyl) amino) pyrimidin-4-yl } [ 1-benzyl (4-piperidinyl) ] amine; {5- (4- (N, N-dimethylamino) phenyl) -2- [ (4-pyridylmethyl) amino) pyrimidin-4-yl- } [ 1-benzyl (4-piperidinyl) ] amine; {5- (phenyl-4-carboxamido) -2- [ (4-picolyl) amino) pyrimidin-4-yl } [ 1-benzyl (4-piperidinyl) ] -amine; {5- (4-carboxyphenyl) -2- [ (4-picolyl) amino) pyrimidin-4-yl } [ 1-benzyl- (-4-piperidinyl) ] amine; {5- (2-thienyl) -2- [ (4-pyridylmethyl) amino) pyrimidin-4-yl } [ 1-benzyl (4-piperidinyl) ] amine; {5- (2-furyl) -2- [ (4-picolyl) amino) pyrimidin-4-yl } [ 1-benzyl (4-piperidinyl) ] amine; {5- (3-furyl) -2- [ (4-picolyl) amino) pyrimidin-4-yl } [ 1-benzyl (4-piperidinyl) ] amine; n (4) - (1-benzyl-piperidin-4-yl) -5- (3-chloro-4-fluoro-phenyl) -N (2) -pyridin-2-ylmethyl-pyrimidine-2, 4-diamine; n- (3- [4- (1-benzyl-piperidin-4-ylamino) -2- [ (pyridin-2-ylmethyl) -amino ] -pyrimidin-5-yl } phenyl) -acetamide; 3- [4- (1-benzyl-piperidin-4-ylamino) -2- [ (pyridin-2-ylmethyl) -amino ] -pyrimidin-5-yl ] -phenol; and 4- {4- (1-benzylpiperidin-4-ylamino) -2- [ (pyridin-2-ylmethyl) -amino ] -pyrimidin-5-yl } N, N-di-methyl-benzamide.

In other embodiments, the small molecule PKC-theta inhibitor is selected from substituted pyridine compounds, as described, for example, by Brunette in U.S. patent application publication US 2006/0217417, which is incorporated herein by reference in its entirety. These compounds are represented by formula (XXIII):

wherein X is a bond or C_1-6Substituted or unsubstituted alkyl, wherein one or two methylene units may be substituted by oxygen or sulfur atoms; y is-NH-, -O-or-S-; r¹Is C_3-6Substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; r²Selected from the following groups: trifluoromethyl, cyano, -CONH₂Halogen and nitro; r³Is that

Wherein p is an integer from 1 to 3, including 1 and 3; q is an integer from 0 to 3, including 0 and 3; n is an integer from 0 to 5, including 0 and 5; r⁴And R⁵Each independently selected from the group consisting of: hydrogen, C_1-6Substituted or unsubstituted alkyl, or wherein R⁴And R⁵Together form a methylene bridge which, together with the nitrogen atom between them, forms a four-to six-membered substituted or unsubstituted ring, in which one methylene group is optionally substituted by an oxygen, sulfur or NR groupWherein R is hydrogen or C_1-6Substituted or unsubstituted alkyl; tautomers and pharmaceutically acceptable salts, solvates, or amino-protected derivatives thereof.

Non-limiting examples of compounds having formula (XXIII) include: 5-nitro-N4-piperidin-4-ylmethyl-N2- (2-trifluoromethoxy-benzyl) -pyridine-2-, 4-diamine; n2- (2, 3-dichloro-benzyl) -5-nitro-N4-piperidin-4-ylmethyl-pyridine-2, 4-diamine; n2- [2- (3-chloro-phenyl) -ethyl ] -5-nitro-N4-piperidin-4-ylmethyl-pyridine-2, 4-diamine; 5-nitro-N2-phenethyl-N4-piperidin-4-ylmethyl-pyridine-2, 4-diamine; n4- (4-aminomethyl-cyclohexylmethyl) -5-nitro-N2- (2-trifluoromethoxy-benzyl-) -pyridine-2, 4-diamine; n4- (4-aminomethyl-cyclohexylmethyl) -N2- (2, 3-dichloro-benzyl) -5-nitro-pyridine-2, 4-diamine; n4- (4-aminomethyl-cyclohexylmethyl) -5-nitro-N2-phenethyl-pyridine-2, 4-diamine; n4- (4-aminomethyl-cyclohexylmethyl) -N2- [2- (3-chloro-phenyl) -ethyl ] -5-nitro-1-pyridine-2, 4-diamine; n4- (4-aminomethyl-cyclohexylmethyl) -5-nitro-N2- (2-chloro-benzyl) -pyridine-2, 4-diamine; n4- (4-trans-aminomethyl-cyclohexylmethyl) -5-nitro-N-2- (2-trifluoromethoxy-benzyl) -pyridine-2, 4-diamine; n4- (4-trans-amino-cyclohexylmethyl) -5-nitro-N2- (2-trifluoromethoxy-benzyl-) -pyridine-2, 4-diamine; 4- [ (4-aminomethyl-cyclohexylmethyl) -amino ] -6- (2-chloro-benzylamino) -nicotinamide; and 4- [ (4-aminomethyl-cyclohexylmethyl) -amino ] -6- (2-chloro-benzylamino) -nicotinonitrile.

In other embodiments, the small molecule PKC-theta inhibitor is selected from indolyl-pyrroledione derivatives, as disclosed, for example, by Auberson in U.S. patent application publication US 2007/0142401, which is incorporated herein by reference in its entirety. These compounds are represented by formula (XXIV):

wherein

R_aIs H; c_1-4An alkyl group; or by OH, NH₂、NHC_1-4Alkyl or N (di-C)_1-4Alkyl radical)₂Substituted C_1-4Alkyl radical；

R_bIs H; or C_1-4An alkyl group;

r is a radical of the formula (a), (b), (c), (d), (e) or (f)

Wherein R is₁、R₄、R₇、R₈、R₁₁And R₁₄Each is OH, SH, a heterocyclic residue, NR₁₆R₁₇Wherein R is₁₆And R₁₇Each independently is H or C_1-4Alkyl, or R₁₆And R₁₇Together with the nitrogen atom to which they are bound form a heterocyclic residue, or of the formula α -X-R_c-Y (α) wherein X is a direct bond, O, S or NR₁₈Wherein R is₁₈Is H or C_1-4Alkyl radical, R_cIs C_1-4Alkylene or C_1-4Alkylene radical, one of which is CH₂Is controlled by CR_xR_yIs substituted in which R_xAnd R_yOne of which is H and the other is CH₃，R_xAnd R_yAre each CH₃Or R_xAnd R_yTogether form-CH₂-CH₂And Y is attached to a terminal carbon atom and is selected from OH, heterocyclic residue and-NR₁₉R₂₀Wherein R is₁₉And R₂₀Each independently is H, C_3-6Cycloalkyl radical, C_3-6cycloalkyl-C_1-4Alkyl, aryl-C_1-4Alkyl or C_1-4Alkyl, optionally substituted on the terminal carbon atom by OH, or R₁₀And R₂₀Together with the nitrogen atom to which they are bound form a heterocyclic residue;

R₂、R₃、R₅、R₆、R₉、R₁₀、R₁₂、R₁₃、R₁₅and R'₁₅Each independently of the other being H, halogen, C_1-4Alkyl, CF₃、OH、SH、NH₂、C_1-4Alkoxy radical, C_1-4Alkylthio, NHC_1-4Alkyl, N (di-C)_1-4Alkyl radical)₂Or CN;

e is-N ═ and G is-CH ═ or E is-CH ═ and G is-N ═ or; and

the ring A is optionally substituted and the ring B is optionally substituted,

or a salt thereof.

In illustrative examples, the heterocyclic residue is R₁、R₄、R₇、R₈、R₁₁、R₁₄Or Y, or each independently of NR₁₆R₁₇Or NR₁₉R₂₀The heterocyclic residue formed is a three-to eight-membered saturated, unsaturated or aromatic heterocyclic ring containing 1 or 2 heteroatoms, and is optionally substituted on one or more ring carbon atoms and/or on a ring nitrogen atom (when present).

In a particular embodiment, the heterocyclic residue is R₁、R₄、R₇、R₈、R₁₁、R₁₄Or Y, or each independently of NR₁₆R₁₇Or NR₁₉R₂₀The heterocyclic residue formed is a residue of formula (γ).

Wherein

Ring D is a 5, 6 or 7 membered saturated, unsaturated or aromatic ring;

X_bis-N-, -C-or-CH-;

X_cis-N ═ NR_f–、–CR_f'or-CHR_f'-, wherein R_fIs a substituent of a ring nitrogen atom and is selected from C_1-6Alkyl, acyl, C_3-6Cycloalkyl radical, C_3-6cycloalkyl-C_1-4Alkyl, phenyl-C_1-4An alkyl group;

heterocyclic residue, and formula β -R₂₁-residue of Y' (β)

Wherein R is₂₁Is C interrupted by O_1-4Alkylene or C_2-4Alkylene and Y' is OH, NH₂、NH(C_1-4Alkyl) or N (C)_1-4Alkyl radical)₂；R_f'Is a ring carbon atomA substituent of a group C_1-4An alkyl group;

C₃-cycloalkyl optionally further substituted by C_1-4-alkyl substitution;

wherein p is 1,2 or 3; CF (compact flash)₃；

Halogen; OH; NH (NH)₂；–CH₂–NH₂；–CH₂-OH; piperidin-1-yl and pyrrolidinyl;

C₁and C₂The bond between is saturated or unsaturated;

C₁and C₂Each independently is a carbon atom, which is optionally substituted on a ring carbon atom with one or two substituents selected from the above; and

C₃and X_bAnd C₁And X_bThe lines in between represent the number of carbon atoms required to obtain a 5-, 6-or 7-membered ring D, respectively.

In other non-limiting examples of compounds according to formula (XXIV)

Ra is H, CH₃、CH₂–CH₃Or an isopropyl group, or a mixture of two or more,

rb is H, halogen, C_1-6-alkoxy, or C_1-6Alkyl, or

I.R is a radical of the formula (a)

Wherein R is₁Is optionally CH-substituted in position 3 or 4₃Substituted piperazin-1-yl; or 4, 7-diaza-spiro (2.5)]Oct-7-yl; r₂Is Cl, Br, CF₃Or CH₃(ii) a And R is₃Is H, CH₃Or CF₃(ii) a When Ra is H or CH₃，R_bIs H, and R₁In the case of 4-methyl-1-piperazinyl, R₃Is not H, or

R is a radical of the formula (b)

Wherein R is₄Is CH substituted in 3 and/or 4 position₃Substituted piperazin-1-yl, or 4, 7-diaza-spiro [2.5]Oct-7-yl; when R is₄In the case of 4-methyl-1-piperazinyl, Ra is other than H or CH₃(ii) a Or

R is a residue of formula (c)

Wherein R is₁₄Is piperazin-1-yl, optionally CH-substituted in the 3-and/or 4-position₃Substituted or in the 3-position by ethyl, phenyl-C_1-4Alkyl radical, C_1-4alkoxy-C_1-4Alkyl or halo-C_1-4Alkyl substitution; or 4, 7-diaza-spiro [2.5 ]]Oct-7-yl; r₁₅Is halogen, CF₃Or CH₃(ii) a When Ra is H or CH₃Rb is H, and R₁₄In the case of 4-methyl-1-piperazinyl, R₁₅Is not CH₃；R₁₆Is H, CH₃Or CF₃(ii) a When R is₁₅Is Cl, Ra is H or CH₃Rb is H and R₁₄In the case of 4-methyl-1-piperazinyl, R₁₆Is not H; or

R is a radical of the formula (d)

Wherein R is₈Is piperazin-1-yl, 3-methylpiperazin-1-yl or 4-benzylpiperazin-1-yl; or

V.R is a radical of the formula (e)

Wherein R is₉Is a 4, 7-diaza-spiro [2.5 ]]Oct-7-yl; or piperazin-1-yl substituted at the 3-position with methyl or ethyl, and optionally substituted at the 4-position with methyl.

In some embodiments of compounds according to formula (XXIV),

when R is represented by the formula (a),

R₁is- (4-methyl-piperazin-1-yl), 1-piperazinyl, 3-methyl-piperazin-1-yl or- (4, 7-diaza-spiro [2.5 ]]Oct-7-yl),

R₂is 2-Cl or 2-CH₃，

R₃Is 3-CH₃、3-CF₃Or a combination of H and a nitrogen atom,

ra is H or CH₃；

And when

When R is represented by the formula (b),

R₄is- (4, 7-diaza-spiro [2.5 ]]Oct-7-yl), 3-methyl-piperazin-1-yl or 4-methyl-3-methyl-piperazin-1-yl,

R_ais H or CH₃；

And when

When R is represented by the formula (c),

R₁₄is-4-methyl-piperazin-1-yl, 3-methyl-piperazin-1-yl, -4, 7-diaza-spiro [2.5]Oct-7-yl, 1-piperazinyl, 4-methyl-3-methyl-piperazin-yl, 3-methoxyethyl-piperazin-1-yl, 3-ethyl-piperazin-1-yl, 3-benzyl-piperazin-1-yl, or 3-CH₂An F-piperazin-1-yl group,

R₁₅is Cl, Br, CF₃、F，

R₁₆Is CH₃、H、CH₂–CH₃，

R_aIs H or CH₃，

R_bIs H, CH₂–CH₂–CH₃、F、CH(CH₃)₂、Cl、OCH₃、CH₃Or CH₂–CH₃；

And when

When R is represented by the formula (d),

R₈is 3-methyl-piperazin-1-yl, 4-benzyl-1-piperazinyl or 1-piperazinyl,

R_ais CH₃Or H;

and when

When R is represented by the formula (e),

R₉is-4, 7-diaza-spiro [2.5 ]]Oct-7-yl, 3-ethyl-piperazin-1-yl, 3-methyl-piperazin-1-yl, 4-methyl-3-methyl-piperazin-1-yl, or 3-ethyl-piperazin-1-yl,

R_ais H, CH₂–CH₃Or CH (CH)₃)₂，

R_bIs CH₃、F、CH(CH₃)₂、OCH₃、CH₂–CH₃Or Cl.

Particular embodiments of compounds according to formula (XXIV) include 3- [ 2-chloro-5- (4-methyl-piperazin-1-yl) -3-trifluoromethyl-phenyl ] -4- (1H-indol-3-yl) -pyrrole-2, 5-dione, which has the following formula:

3- (1H-indol-3-yl) -4- [2- (4-methyl-piperazin-1-yl) -quinazolin-4-yl ] -pyrrole-2, 5-dione having the formula:

in other embodiments, the PKC-theta inhibitor is selected from the group consisting of selective PKC-theta small molecule compounds disclosed by Ajioka in U.S. patent application publication 2013/0225687, which is incorporated herein by reference in its entirety. These compounds are represented by formula (XXV):

wherein:

y is selected from-O-and-S-; and

each R^lIndependently selected from n-propyl, isopropyl, tert-butyl, tert-amyl, CF₃、OCF₃Cyclopentyl, pyrrolyl and CO₂H, and salts, hydrates, and prodrugs thereof, thereby selectively inhibiting PKC-theta.

In a preferred embodiment, the PKC-theta inhibitor is an inhibitor of nuclear translocation/localization of PKC-theta. Representative inhibitors of this type include those disclosed by Rao et al in international publication No. WO 2017/132728a1, which is incorporated herein by reference in its entirety. These compounds are protein molecules represented by the formula (XXVI):

Z₁X₁X₂X₃X₄IDX₅PPX₆X₇X₈X₉X₁₀X₁₁Z₂(XXVI)

wherein:

“Z₁"and" Z₂"independently deleted or independently selected from at least one protein moiety comprising from about 1 to about 50 amino acid residues (and all integer amino acid residues therebetween) and a protecting moiety;

“X₁"deleted or selected from the group consisting of R, K and modified forms thereof;

“X₂"and" X₃"is independently selected from the group consisting of basic amino acid residues, including R, K and modified forms thereof;

“X₄"selected from charged amino acid residues, including R, K, D, E and modified forms thereof;

“X₅"deleted or is W or modified form thereof;

“X₆"selected from aromatic or basic amino acid residues, including F, Y, W, R, K and modified forms thereof;

“X₇"selected from the group consisting of basic amino acid residues, including R, K and modified forms thereof;

“X₈"deleted or is P or modified form thereof;

“X₉"selected from the group consisting of basic amino acid residues, including R, K and modified forms thereof;

“X₁₀"is selected from the group consisting of hydrophobic residues, including V, L, I, M and modified forms thereof and P and modified forms thereof;

“X₁₁"is selected from the group consisting of basic amino acid residues, including R, K and modified forms thereof.

In some casesIn an embodiment, "X₁"to" X₁₁"selected from the group consisting of one or more of:

“X₁"deleted or is R;

“X₂"is R;

“X₃"is K;

“X₄"is E or R;

“X₅"deleted or is W;

“X₆"is F or R;

“X₇"is R;

“X₈"deleted or is P;

“X₉"is K;

“X₁₀"is V or P; and

“X₁₁"is K.

In some embodiments, "Z" is₁"consists of 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 amino acid residue. In some embodiments, "Z" is₂"consists of 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 amino acid residue. In some embodiments, "Z" is₁"and" Z₂The amino acid residue in "is selected from any amino acid residue.

In some embodiments, "Z" is₁"is a protein molecule represented by formula XXVII:

X₁₂X₁₃X₁₄X₁₅X₁₆(XXVII)

wherein:

“X₁₂"deleted or protected;

“X₁₃"deleted or selected from P and basic amino acid residues, including R, K and modified forms thereof;

“X₁₄"deleted or selected from P and basic amino acid residues, including R, K and modified forms thereof;

“X₁₅"deleted or selected from P and basic amino acid residues, including R, K and modified forms thereof;

“X₁₆"deleted or selected from P and basic ammoniaAmino acid residues, including R, K and modified forms thereof.

In some embodiments, "Z" is₂"is a protein molecule represented by formula XXVIII:

X₁₇X₁₈X₁₉X₂₀(XXVIII)

wherein:

“X₁₇"deleted or selected from any amino acid residue;

“X₁₈"deleted or selected from any amino acid residue;

“X₁₉"deleted or selected from any amino acid residue;

“X₂₀"deleted or protected.

In some embodiments, "Z" is₁"and" Z₂"deletion".

In a specific embodiment, the protein molecule of formula XXVI comprises SEQ ID NO: 4 or 5, consisting or consisting essentially of:

RKEIDPPFRPKVK[SEQ ID NO：4]

RRKRIDWPPRRKPK[SEQ ID NO：5]。

SEQ ID NO: 4 and SEQ ID NO 5 are referred to as "importib 4759" and "importib 4759-O1", respectively, in WO 2017/132728A 1.

In some embodiments of the proteinaceous molecule according to formula (XXVI), the molecule comprises at least one membrane penetrating moiety. The membrane-penetrating moiety may be conjugated at any point of the protein molecule. Suitable membrane-penetrating moieties include lipid moieties, cholesterol, and proteins, such as cell-penetrating peptides and polycationic peptides; especially the lipid fraction.

Non-limiting examples of cell penetrating peptides include, for example, peptides described in US 20090047272, US 20150266935, and US 20130136742. Thus, suitable cell penetrating peptides may include, but are not limited to, basic poly (Arg) and poly (Lys) peptides, as well as basic poly (Arg) and (Lys) peptides that contain non-natural analogs of Arg and Lys residues, such as YGRKKRPQRRR (HIV TAT)_47-57)、RRWRRWWRRWWRRWRR(W/R)、CWK₁₈(AlkCWK₁₈)、K₁₈WCCWK₁₈(Di-CWK₁₈)、WTLNSAGYLLGKINLKALAALAKKIL(Transportan)、GLFEALEELWEAK(DipaLytic)、K₁₆GGCRGDMFGCAK₁₆RGD(K₁₆RGD)、K₁₆GGCMFGCGG(P1)、K₁₆ICRRARGDNPDDRCT(P2)、KKWKMRRNQFWVKVQRbAK(B)bA(P3)、VAYISRGGVSTYYSDTVKGRFTRQKYNKRA(P3a)、IGRIDPANGKTKYAPKFQDKATRSNYYGNSPS(P9.3)、KETWWETWWTEWSQPKKKRKV(Pep-1)、PLAEIDGIELTY(Plae)、K₁₆GGPLAEIDGIELGA(Kplae)、K₁₆GGPLAEIDGIELCA(cKplae)、GALFLGFLGGAAGSTMGAWSQPKSKRKV(MGP)、WEAK(LAKA)₂-LAKH(LAKA)₂LKAC(HA2)、(LARL)₆NHCH₃(LARL4₆)、KLLKLLLKLWLLKLLL(Hel-11-7)、(KKKK)₂GGC(KK)、(KWKK)₂GCC(KWK)、(RWRR)₂GGC (RWR), PKKKRKV (SV40NLS7), PEVKKKRKPEYP (NLS12), TPPKKKRKVEDP (NLS12a), GGGGPKKKRKVGG (SV40NLS13), GGGFSTSLRARKA (AV NLS13), CKKKKKKSEDEYPYVPN (AV RMENLS17), CKKKKKKKSEDEYPYVPNFSTSLRARKA (AV FP NLS28), LVRKKRKTEEESPLKDKDAKKSKQE (SV40N1NLS24), and K₉K₂K₄K₈GGK₅(Loligomer); HSV-1 envelope protein (regulatory protein) VP 22; HSV-1 envelope protein VP22r fused to the Nuclear Export Signal (NES); mutant B subunit of escherichia coli enterotoxin EtxB (H57S); detoxified exotoxin a (eta); the protein transduction domain GRKKRRQRRRRPPQ of HIV-1Tat protein; drosophila melanogaster (Drosophila melanogaster) antennal domain Antp (amino acids 43-58), RQIKIWFQNRRMKWKK; buforin ii, trssrraglqfpvgrvhrllrk; hClock- (amino acids 35-47) (human Clock protein DNA binding peptide), KRVSRNKSEKKRR; MAP (amphiphilic peptide-like), KLALKLALKALKAALKLA; K-FGF, AAVALLPAVLLALLAP; a Ku 70-derived peptide comprising a peptide selected from VPMLKE, VPMLK, PMLKE or PMLK; prion, mouse Prpe (amino acids 1-28), MANLGYWLLALFVTMWTDVGLCKKRPKP; pVEC, llilrrrirkqahahsk; Pep-I, KETWWETWWTEWSQPKKKRKV; SynBl, RGGRLSYSRRRFSTSTTGR; transportan, GWTLNSAGYLLGKINLKALAALAKKIL; transportan-10, AGYLLGKINLKALAALAKKIL; CADY, Ac-GLWRALWRLLRSLWRLLWRA-cysteamine; pep-7, SDLWEMMMVSLACQY; HN-1, TSPLNIHNGQKL; VT5, DPKGDPKGVTVTVTVTVTGKGDPKPD or pISL, RVIRVWFQNKRCKDKK。

In a preferred embodiment, the membrane penetrating moiety is a lipid moiety, e.g. C₁₀-C₂₀Fatty acyl, especially stearyl (C)₁₈) Hexadecanoyl (palmitoyl; c₁₆) Or tetradecanoyl (myristoyl; c₁₄) (ii) a Most particularly tetradecanoyl. In preferred embodiments, the membrane permeable moiety is conjugated to the N-or C-terminal amino acid residue of the protein molecule, or through an amine of a lysine side chain, in particular to the N-terminal amino acid residue of the protein moiety.

2.2PD-1 binding antagonists

A PD-1 binding antagonist is suitably a molecule that inhibits signaling by PD-1, and includes molecules that inhibit the binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. The antagonist may be an antibody, immunoadhesin, fusion protein or oligopeptide.

The PD-1 binding antagonist is preferably an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab, OPDIVO), Merck3475 (MK-3475, pembrolizumab, KEYTRUDA), CT-011 (pidilizumab), MEDI-4736 (DULUVACUMAB) MEDI-0680(AMP-514), PDR001, REGN2810, BGB-108, and BGB-A317. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nituzumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 and

is an anti-PD-1 antibody described in WO 2006/121168. Pembrolizumab, also known as MK-3475, Merck3475, Lamborlizumab,

And SCH-900475 at WAn anti-PD-1 antibody described in O2009/114335. CT-011, also known as hBAT, hBAT-1 or pidemizumab, is an anti-PD-1 antibody described in WO 2009/101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc soluble fusion receptor described in WO2010/027827WO 2011/066342.

In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4). In another embodiment, there is provided an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7, or a light chain variable region amino acid sequence. In another embodiment, an isolated anti-PD-1 antibody is provided that comprises heavy and/or light chain sequences, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence of seq id no:

or (b) the light chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a light chain sequence that is:

in some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS registry number: 1374853-91-4). In another embodiment, there is provided an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, or a light chain variable region amino acid sequence of seq id no. In another embodiment, an isolated anti-PD-1 antibody is provided that comprises heavy and/or light chain sequences, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence of seq id no:

or (b) the light chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a light chain sequence that is:

the invention also encompasses antibody fragments comprising the heavy and light chain HVRs of a full-length anti-PD-1 antagonist antibody.

In another aspect, provided herein is a nucleic acid encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expressing a nucleic acid encoding any of the previously described anti-PDL 1, anti-PD-1, or anti-PDL 2 antibodies. In yet another specific aspect, the vector further comprises a host cell suitable for expression of the nucleic acid. In yet another specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In a further specific aspect, the eukaryotic cell is a mammalian cell, such as Chinese Hamster Ovary (CHO).

The antibody or antigen-binding fragment thereof can be prepared using methods known in the art, for example, by a method comprising culturing a host cell containing a nucleic acid encoding any of the foregoing anti-PD-1 or antigen-binding fragments in a form suitable for expression under conditions suitable for production of such antibody or fragment, and recovering the antibody or fragment.

In some embodiments, the isolated anti-PD-1 antibody is aglycosylated. Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid (most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used). The glycosylation sites can be conveniently removed from the antibody by altering the amino acid sequence to remove one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Changes may be made by substituting asparagine, serine or threonine residues within the glycosylation site with other amino acid residues (e.g., glycine, alanine or conservative substitutions).

2.3 adjuvants

In some embodiments, the PKC-theta inhibitor and the PD-1 binding antagonist are administered concurrently with an adjuvant to treat or adjunctively treat the T cell dysfunctional disorder. Non-limiting examples of adjuvants include cytotoxic agents, gene therapy agents, DNA therapy agents, viral therapy agents, RNA therapy agents, immunotherapeutic agents, bone marrow transplantation agents, nanotherapeutic agents, or combinations of the foregoing. The adjuvant may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the adjuvant is a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the adjuvant is a side-effect limiting agent (e.g., an agent intended to reduce the occurrence and/or severity of a therapeutic side effect, such as an anti-nausea agent, etc.). In some embodiments, the adjuvant is a radiotherapeutic agent. In some embodiments, the adjuvant is an agent that targets the PI3K/AKT/mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventive agent. In some embodiments, the adjuvant is an immunotherapeutic agent, e.g., a blocking antibody, Yipriomamab (also known as MDX-010, MDX-101, or MDX-101)

) Tramelizumab (tremelimumab) (also known as ticilimumab or CP-675,206), antagonists directed against B7-H3 (also known as CD276), e.g., blocking antibody MGA271, antagonists against TGF- β, e.g., meridamycin antibody (also known as CAT-192), frasuzumab (also known as GC1008) or LY2157299, T cells expressing a Chimeric Antigen Receptor (CAR) (e.g., cytotoxic T cells or CTLs), T cells comprising a dominant negative TGF- β receptor (e.g., a dominant negative TGF- β type II receptor), agonists against CD137 (also known as TNFRSF9, 4-1BB or ILA), e.g., the activated antibody Uribritumomab (also known as BMS-663513), agonists against CD40, e.g., the activated antibody CP-8793, agonists against 40 (also known as CD134), e.g., activated antibody administered in combination with anti-tamsultrine 2 antibody (e.g., Agonon), agonists against CD40, e antibody, e-m-T-0603, e-T-co-T-463, or, e

Genentech), DMUC5754A, antibody-drug conjugates targeting endothelin B receptor (EDNBR), e.g., antibodies against EDNBR conjugated with MMAE, angiogenesis inhibitors, antibodies against VEGF, e.g., VEGF-a, bevacizumab (also known as VEGF-a), bevacizumab

Genentech), antibodies against angiopoietin 2 (also known as Ang2), MEDI3617, antineoplastic agents, agents targeting CSF-1R (also known as M-CSFR or CD115), anti-CSF-1R (also known as IMC-CS4), interferons, such as IFN- α or IFN- γ, Roferon-A, GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhuGM-CSF, sargramostim or

) IL-2 (also known as aldesleukin or

) IL-12, antibodies targeting CD20 (in certain embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or GA 20)

) Or rituximab), an antibody targeting GITR (in certain embodiments, the antibody targeting GITR is TRX518), in combination with a cancer vaccine (in certain embodiments, the cancer vaccine is a peptide cancer vaccine, in some embodiments an individualized peptide vaccine; in some embodiments, the peptide Cancer vaccine is a multivalent long peptide, polypeptide, peptide mixture, hybrid peptide, or peptide pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci, 104: 14-21,2013)), in combination with an adjuvant, TLR agonist, e.g., Poly-ICLC (also known as PolyiCCL)

) LPS, MPL, or CpG ODN, TNF- α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an HVEM antagonist, an ICOS agonist, for example, by administering ICOS-L, or an agonist antibody directed against ICOS, an agent targeting CX3CL1, an agent targeting CXCL10, an agent targeting CCL5, an LFA-1 or ICAM1 agonist, a selectin agonist, a targeted therapeutic, an inhibitor of B-Raf, Verofibrib (also known as CCL 5)

) Dabrafenib (also known as delofibri)

) Erlotinib (also known as

) MEK inhibitors, for example inhibitors of MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2), cobicistinib (also known as GDC-0973 or XL-518), trametinib (also known as GDC-0973 or XL-518)

) K-Ras inhibitors, c-Met inhibitors, entizumab (onartuzumab, also known as MetMAb), Alk inhibitors, AF802 (also known as CH5424802 or alectinib), inhibitors of phosphatidylinositol 3 kinase (PI3K), BKM120, idelizib (also known as GS-1101 or CAL-101), periplosine (also known as KRX-0401), Akt, MK2206, GSK 066993, GDC-0941, inhibitors of mTOR, sirolimus (also known as rapamycin), temsirolimus (also known as CCI-779 or alectinib)

) Everolimus (also known as RAD001), ridaforolimus (also known as AP-23573, MK-8669 or deforolimus), OSI-027, AZD8055, INK128, dual PI3K/mTOR inhibitors, XL765, GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-04691502, PF-05212384 (also known as PKI-587). The adjuvant may be one or more cytotoxic or chemotherapeutic agents described herein.

In some embodiments, the adjuvant is an anti-infective drug. The anti-infective drug is suitably selected from antimicrobial agents including, but not limited to, compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeasts, fungi, protozoa, and the like, and thus includes antibiotics, anti-amebiasis, antifungals, antiprotozoals, antimalarials, antituberculosis drugs, and antiviral drugs. Anti-infective agents also include within their scope anthelmintics and nematicides. Illustrative antibiotics include quinolones (e.g., amifloxacin, cinofloxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, rosoxaxin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin and galaxacin), tetracyclines, glycylcyclines and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, limacycline, mecycline, oxytetracycline, tetracycline, tigecycline, linezolid, epothizole (epozolid)), glycopeptides, aminoglycosides (e.g., kanamycin, abercacin, brevicacin, gentamicin, kanamycin, meomin, mecomycin, dibekacin, gentamicin, kanamycin, Netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), beta-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, ceftriazine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefotaxime, cefimidazole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftriaxone, cefuroxime, cefofurin, cefozopran, cephalosporin (cephacetrile), cephalexin, cefalexin, ceftiofur, cephaloridine, cephalosporin, amoxicillin, doxorubicin, cephalosporin, ceftibufenacin, cephalosporin, ceftriaxone, latamoxef, amixiline, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carbenicillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sunitinil, temocillin, ticarcillin, cephadrine, SC004, KY-020, cefdinir, cefotiazem, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, ceftizolid, ME1228, KP-736, CP-6232, Ro09-1227, OPC-20000, 206LY 763), rifamycins, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, romycin, roxamikacin, roxithromycin, oleandomycin), ketoesters (e.g., telithromycin), and ketoesters (e.g., telithromycin, Quinomycin), coumaromycin, lincosamines (e.g., clindamycin, lincomycin), and chloramphenicol. Exemplary antiviral agents include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivison sodium, foscarnet, ganciclovir, indinavir sulfate, lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir and zivudine. Non-limiting examples of anti-amebic or anti-protozoal agents include atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole hydrochloride, and pentamidine isethionate. The anthelmintic may be at least one selected from the group consisting of mebendazole, pyrrolidinate, albendazole, ivermectin, and thiabendazole. Exemplary antifungal agents may be selected from amphotericin B, amphotericin B cholesterol sulfate complex, amphotericin B lipid complex, amphotericin B liposome, fluconazole, flucytosine, griseofulvin microparticle, griseofulvin flavone ultramicroparticle, itraconazole, ketoconazole, nystatin, and terbinafine hydrochloride. Non-limiting examples of antimalarial drugs include chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine and pyrimethamine with sulfadoxine. Antituberculosis agents include, but are not limited to, clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.

3. Pharmaceutical compositions and formulations

Also provided herein are pharmaceutical compositions and formulations comprising a PKC-theta inhibitor, a PD-1 binding antagonist, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions and formulations further comprise an adjuvant as, for example, described herein.

Pharmaceutical compositions and formulations as described herein may be prepared by mixing an active ingredient (e.g., a small molecule, nucleic acid or polypeptide) of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's pharmaceutical Sciences 16 th edition, Osol, a. editor (1980))). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g. octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol alcohol; benzalkonium chloride; phenol alcohol,Butanol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (r) ((r))

Baxter International, Inc.). Certain exemplary shasegps and methods of use are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968, including rHuPH 20. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

In some embodiments, particularly with respect to peptide and polypeptide active agents (e.g., antibodies, inhibitory peptides, and immunoadhesins), the active agent and optionally a pharmaceutically acceptable carrier are in the form of a lyophilized formulation or an aqueous solution. Exemplary lyophilized antibody formulations are described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including histidine-acetate buffer.

The compositions and formulations herein may also contain other active ingredients necessary for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

The active ingredient may be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol,

A. editing (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Formulations for in vivo administration are generally sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

The formulations may be administered systemically or locally depending on the particular condition being treated. Suitable routes may, for example, include oral, rectal, transmucosal or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Formulations and application techniques can be found in the latest edition of "Remington's pharmaceutical Sciences," Mack Publishing Co., Easton, Pa..

4. Therapeutic uses

The present invention discloses that PKC-theta inhibitors and PD-1 binding antagonists (also referred to herein as "therapeutic combinations" or "combination therapies") are useful for treating T cell dysfunctional disorders, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection in an individual. In particular embodiments, therapeutic combinations are disclosed for treating or delaying the progression of cancer, including metastatic cancer, and for preventing cancer recurrence. In this regard, any PKC-theta inhibitor and PD-1 binding antagonist known in the art or described herein may be used.

In some embodiments, the combination therapy further comprises the use or administration of an adjuvant (e.g., a chemotherapeutic agent) as, for example, described herein.

Suitably, the individual to be treated with the combination therapy comprises T cells having a mesenchymal phenotype (e.g. CD 8)⁺T cells) that suitably exhibit T cell fatigue or anergy, and in representative examples of this type, express EOMES at levels greater than TBET levels and/or have increased PD-1 expression, in some embodiments, T cells have impaired or suppressed immune function, and suitably express reduced biomarkers of T cell activation (e.g., reduced production and/or secretion of cytokines such as II-2, IFN- γ, and TNF- α), hi these embodiments, T cells suitably express ZEB1 at levels greater than the TBET levels expressed in the same T cells, or greater than the b1 levels expressed in the nucleus of activated T cells, this level of ZEB1 is also referred to herein as a PKC-T-receptor, a ZEB-receptor, a ZEB-receptor, a ZEB1 receptor, and a zet cell receptor immune receptor for which the T cells express nuclear PKC- θ levels greater than TBET levels expressed in the same T cells, and/or greater than the expression levels in activated T cells.

In some embodiments, the subject is a human.

In some embodiments, the individual has been treated with the PD-1 binding antagonist prior to combination treatment with the PD-1 binding antagonist and a PKC-theta inhibitor (e.g., a nuclear translocation inhibitor of PKC-theta).

In some embodiments, the individual has a cancer that is resistant to one or more PD-1 binding antagonists (has been demonstrated to be resistant). In some embodiments, the tolerance to the PD-1 antagonist comprises relapse of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer at the original site or new site after treatment. In some embodiments, tolerance to a PD-1 binding antagonist includes progression of cancer during treatment with the PD-1 binding antagonist. In some embodiments, the tolerance to the PD-1 binding antagonist comprises a cancer that is not responsive to treatment. The cancer may become resistant at the beginning of the treatment or become resistant during the treatment. In some embodiments, the cancer is at an early or late stage.

In some embodiments of any of the methods, assays, and/or kits, any one or more T cell functional biomarkers are detected in a sample using a method selected from the group consisting of: FACS, western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH and combinations thereof.

In some embodiments of any of the methods, assays, and/or kits, any one or more T cell functional biomarkers are detected in a sample by protein expression. In some embodiments, protein expression is determined by Immunohistochemistry (IHC). In some embodiments, any one or more of the T cell functional biomarkers is detected using an antibody that specifically binds to the respective biomarker. In some embodiments, for example, a nuclear PKC-theta and/or ZEB1 biomarker is detected in the nucleus of a T cell using IHC. In some embodiments, a complex comprising nuclear PKC-theta and ZEB1 biomarkers is detected in the nucleus of a T cell.

In some embodiments, the combination therapies of the invention comprise administering a PKC-theta inhibitor and a PD-1 binding antagonist. The PKC-theta inhibitor and PD-1 binding antagonist may be administered in any suitable manner known in the art. For example, the PKC-theta inhibitor and the PD-1 binding antagonist may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the PKC-theta inhibitor and the PD-1 binding antagonist are in separate compositions. In some embodiments, the PKC-theta inhibitor is in the same composition as the PD-1 binding antagonist. Thus, the combination therapy may comprise administration of a PKC-theta inhibitor separately, simultaneously or sequentially with a PD-1 binding antagonist. In some embodiments, this may be achieved by administering a single composition or pharmaceutical formulation comprising both types of agents, or by administering two separate compositions or formulations at the same time, wherein one composition comprises a PKC-theta inhibitor and the other comprises a PD-1 binding antagonist. In other embodiments, treatment with a PKC-theta inhibitor may be preceded or followed by treatment with a PD-1 binding antagonist at intervals ranging from minutes to days. In embodiments where the PKC-theta inhibitor is used separately from the PD-1 binding antagonist, it will generally be ensured that there is no significant time interval expired between each delivery such that the PKC-theta inhibitor is still able to exert a beneficial effect on the functionally inhibited T cells (e.g., mesenchymal T cells), as described above, particularly to confer T cells with enhanced immune function, including susceptibility of the T cells to reactivation of the PD-1 binding antagonist. In such a case, it is contemplated that the two ingredients will be administered within about 1-12 hours of each other, and more suitably within about 2-6 hours of each other. In some cases it may be desirable to significantly extend the treatment time, however, the time delay between administrations (lapse) is from several hours (2,3,4,5, 6 or 7) to several days (1, 2,3,4,5, 6,7 or 8).

It is contemplated that more than one administration of a PKC-theta inhibitor or PD-1 binding antagonist may be required. Various combinations may be employed, where the PKC-theta inhibitor is "a" and the PD-1 binding antagonist is "B", as shown below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B。

the PKC-theta inhibitor and the PD-1 binding antagonist may be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the PKC- Θ inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist can be administered to prevent or treat a disease. Suitable dosages of the PKC-theta inhibitor and the PD-1 binding antagonist may be determined according to the type of condition being treated, the type of PKC-theta inhibitor and the PD-1 binding antagonist, the severity and course of the condition, the clinical condition of the individual, the clinical history and response to treatment of the individual, and the judgment of the attending physician. In some embodiments, the combination treatment with a PKC-theta inhibitor (e.g., a nuclear translocation inhibitor of PKC-theta) and a PD-1 binding antagonist (e.g., an anti-PD-1 antibody) is synergistic, whereby the effective dose of the PD-1 binding antagonist (e.g., an anti-PD-1 antibody) in the combination is reduced relative to the effective dose of the PD-1 binding antagonist (e.g., an anti-PD-1 antibody) as a single agent.

As a general proposition, a therapeutically effective amount of a peptide or polypeptide active agent (e.g., antibody, peptide inhibitor, immunoadhesin, etc.) administered to a human is from about 0.01 to about 50mg/kg of patient body weight, whether administered one or more times. In some embodiments, the antibody used is about 0.01 to about 45mg/kg, about 0.01 to about 40mg/kg, about 0.01 to about 35mg/kg, about 0.01 to about 30mg/kg, about 0.01 to about 25mg/kg, about 0.01 to about 20mg/kg, about 0.01 to about 15mg/kg, about 0.01 to about 10mg/kg, about 0.01 to about 5mg/kg, or about 0.01 to about 1mg/kg, e.g., administered daily. In some embodiments, a peptide or polypeptide active agent (e.g., an antibody, peptide inhibitor, immunoadhesin, etc.) is administered at 15 mg/kg. However, other dosage regimens may be useful. In one embodiment, the anti-PDL 1 antibody described herein is administered to a human at a dose of about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, or about 1400mg on day 1 of a 21-day cycle. The dose may be administered in a single dose or in multiple doses (e.g. 2 or 3 doses), for example as an infusion. The dose of peptide or polypeptide active agent (e.g., antibody, peptide inhibitor, immunoadhesin, etc.) administered in a combination therapy can be reduced as compared to a single treatment. The progress of the therapy can be readily monitored by conventional techniques.

Small molecule compounds are typically administered at an initial dose of about 0.0001mg/kg to about 1000mg/kg per day. The daily dosage range that may be used is from about 0.01mg/kg to about 500mg/kg, or from about 0.1mg/kg to about 200mg/kg, or from about 1mg/kg to about 100mg/kg, or from about 10mg/kg to about 50 mg/kg. However, the dosage may vary depending on the requirements of the patient, the severity of the condition being treated and the compound being used.

In any event, the dosage may be determined empirically based on the type and stage of disease diagnosed in a particular patient. In the context of the present invention, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the presence, nature and extent of any adverse side effects associated with the administration of a particular compound in a particular patient. Determining the appropriate dosage for a particular situation is within the skill of one in the art. Typically, treatment is initiated at a smaller dose than the optimal dose of the compound. Thereafter, the dosage is increased in small increments until the optimum effect under the particular circumstances is achieved. For convenience, the total daily dose may be divided and administered in portions throughout the day, if desired. The dosage may be administered daily or every other day, as determined by the treating physician. The dosage may also be administered periodically or continuously over a longer period of time (weeks, months or years), for example by use of a subcutaneous capsule, capsule or reservoir, or by patch or pump. In some embodiments, the PKC-theta inhibitor, the PD-1 binding antagonist, and the optional adjuvant (e.g., chemotherapeutic agent) are administered on a routine schedule. Alternatively, combination therapy may be administered at the time of the onset of symptoms.

As used herein, a "routine schedule" refers to a predetermined specified time period. The routine schedule may include time periods of the same or different lengths, so long as the schedule is predetermined. For example, a routine schedule may include administration of a PKC-theta inhibitor, a PD-1 binding antagonist, and optional adjuvants on a daily, every two-day, every three-day, every four-day, every five-day, every six-day, weekly, monthly, or any set number of days or weeks in between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc. Alternatively, the predetermined routine schedule may include administering the PKC-theta inhibitor, the PD-1 binding antagonist, and the optional adjuvant simultaneously on each day of the first week, on each month of the following months, and then every three months. Routine schedules will encompass any particular combination as long as the appropriate schedule including administration on a certain day is determined in advance.

In some embodiments, the methods of treatment and uses may further comprise additional therapies. The additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma radiation.

The efficacy of any of the methods described herein (e.g., combination therapy comprising administering an effective amount of a combination of a PKC-theta inhibitor, a PD-1 binding antagonist, and an optional adjuvant) can be tested in various models known in the art, such as clinical or preclinical models. Suitable preclinical models are exemplified herein, and may further include, but are not limited to, cancer models of ID8 ovarian cancer, GEM model, B16 melanoma, RENCA renal cell carcinoma, CT26 colorectal cancer, MC38 colorectal cancer, and Cloudman melanoma.

The efficacy of any of the methods described herein (e.g., a combination therapy comprising administering an effective amount of a combination of a PKC-theta inhibitor, a PD-1 binding antagonist, and an optional adjuvant) can be tested in establishing a GEM model of a tumor, including but not limited to a non-small cell lung cancer, pancreatic ductal adenocarcinoma, or melanoma. For example, Jackson et al (2001Genes Dev.15 (24): 3243-8) (Kras is described)^G12D) And Lee et al (2012dis. model mech.5 (3): 397-402) (FRT-mediated p53^nullAllele) as described in p53 after treatment with adenovirus recombinase^nullExpression of Kras in background^G12DOf mice, canUsed as a preclinical model for non-small cell lung cancer. As another example, Jackson et al (2001, supra) describe Kras^G12D) And Aguirre et al (2003Genes Dev.17 (24): 3112-26) (p16/p19^nullAllele) of said at p16/p19^nullExpression of Kras in background^G12DCan be used as a preclinical model of Pancreatic Ductal Adenocarcinoma (PDAC). As another example, Dankort et al (2007Genes Dev.21 (4): 379-84) (Braf is described)^V600E) And Trotman et al (2003PLoS biol.1 (3): E59) (PTEN)^nullAllele) after treatment with an inducible (e.g., 4-OHT-treated) recombinase as described in (e.g., 4-OHT), in melanocyte-specific PTEN^nullHaving expression of Braf in the background^V600EThe mouse of melanocytes of (1), which can be used as a preclinical model of melanoma. For any of these exemplary models, after tumor establishment, mice were randomly recruited to treatment groups receiving a combination of PKC-theta inhibitor, PD-1 binding antagonist, and optional adjuvant therapy or control treatment. Tumor size (e.g., tumor volume) is measured during treatment, and overall survival is also monitored.

In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a patient cancer sample as examined using a diagnostic test, as described in the examples herein) comprises Tumor Infiltrating Lymphocytes (TILs), wherein the TILs are within or associated with the cancer tissue. In these embodiments, the expression of any one or more of the T cell function biomarkers disclosed herein in the TIL is assessed. For example, nuclear PKC-theta and ZEB1 can be used as biomarkers of mesenchymal phenotype and T cell activation. Furthermore, TBET, PD-1 and EOMES can be used as biomarkers of T-cell exhaustion characterized by, for example, high levels of inhibitory co-receptors and lacking the ability to produce effector cytokines (where, E.J.2011Nature immunology 12: 492-.

In some embodiments of the methods of the present disclosure, the subject has T cell dysfunction, manifested as a T cell dysfunction disorder. T cell dysfunctional disorders may be characterized by unresponsive or reduced secretion by T cellsCytokines, ability to proliferate or perform cytolytic activity. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by suppression of T cell immune function. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by T cells of a mesenchymal phenotype. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by T cell exhaustion. In some embodiments of the methods of the present disclosure, the T cell is CD4⁺And/or CD8⁺According to the invention, PKC-theta inhibitor treatment may increase expression and effector capacity of biomarkers of T-cell activation (e.g., IL-2, IFN-gamma, and TNF- α), decrease expression of biomarkers of T-cell effect suppression and cancer progression (e.g., ZEB1), decrease expression of biomarkers of T-cell exhaustion (e.g., PD-1 and EOMES), and/or increase expression of the transcription factor TBET that increases IFN-gamma production in cells of the adaptive and innate immune systems⁺T cell, CD8⁺T cells, memory T cells) are induced, activated and/or propagated. In some embodiments, the T cell is CD4⁺And/or CD8⁺T cells.

In some embodiments of the methods of the present disclosure, activated CD4 in an individual⁺And/or CD8⁺T cells are characterized by IFN-gamma producing CD4 compared to prior to administration of the combination⁺And/or CD8⁺Enhanced T cell and/or cytolytic activity. IFN- γ can be measured by any means known in the art, including, for example, Intracellular Cytokine Staining (ICS), which includes cell fixation, permeabilization, and staining with antibodies to IFN- γ. Cytolytic activity can be measured by any means known in the art, for example, a cell killing assay using a mixture of effector and target cells.

In some embodiments, CD8⁺T cells are characterized, for example, by the presence of CD8b expressionIn some embodiments, CD8 is expressed in (e.g., by RT-PCR using, e.g., Fluidigm) (Cd8b also known as the T cell surface glycoprotein CD8 β chain; CD8 antigen; α polypeptide p3' 7; accession number NM-172213)⁺T cells are from peripheral blood. In some embodiments, CD8⁺T cells are from tumors.

In some embodiments, the Treg cells are characterized, for example, by the presence of Fox3P expression (e.g., by RT-PCR, e.g., using Fluidigm) (Foxp3 also known as Forkhead box protein P3; scurfin; Foxp3delta 7; immunodeficiency, polyendocrine, enteropathy, X-linked; accession No. NM _ 014009). In some embodiments, the tregs are from peripheral blood. In some embodiments, the Treg cells are from a tumor.

In some embodiments, the inflammatory or activated T cell is characterized, for example, by the presence of TBET and/or CXCR3 expression, or TBET: EOMES ratio (e.g., by RT-PCR using, for example, Fluidigm). In some embodiments, the inflammatory or activated T cells are from peripheral blood. In some embodiments, the inflammatory or activated T cells are from a tumor.

In some embodiments of the methods of the present disclosure, CD4⁺And/or CD8⁺T cells exhibit an increase in cytokine release selected from IFN- γ, TNF- α, and interleukins such as IL-2 cytokine release can be measured by any means known in the art, for example, using Western blot, ELISA, or immunohistochemical assays to detect the presence of CD4⁺And/or CD8⁺Presence of cytokines released in a sample of T cells.

In some embodiments of the methods of the present disclosure, CD4⁺And/or CD8⁺The T cells are effector memory T cells. In some embodiments of the methods of the present disclosure, CD4⁺And/or CD8⁺Effector memory T cells are characterized by having CD44^{Height of}CD62L^{Is low in}Expression of (2). CD44^{Height of}CD62L^{Is low in}Can be detected by any method known in the art, e.g., by preparing a single cell suspension of the tissue (e.g., cancer tissue) and using a quotient for CD44 and CD62LAntibodies were used for surface staining and flow cytometry. In some embodiments of the methods of the present disclosure, CD4⁺And/or CD8⁺Effector memory T cells are characterized by having expression of CXC R3 (also known as C-X-C chemokine receptor type 3; Mig receptor; IP10 receptor; G protein coupled receptor 9; interferon inducible protein 10 receptor; accession No. NM-001504). In some embodiments, CD4⁺And/or CD8⁺Effector memory T cells are from peripheral blood. In some embodiments, CD4⁺And/or CD8⁺Effector memory T cells are from tumors.

In some embodiments of the methods of the present disclosure, administering to the individual an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist, and optionally an adjuvant, is characterized by an increase in CD8 as compared to prior to administration of the combination therapy⁺Levels of inflammatory markers (e.g., CXCR3) on T cells. CXCR3/CD8⁺T cells can be measured by any method known in the art. In some embodiments, CXCR3/CD8⁺T cells are from peripheral blood. In some embodiments, CXCR3/CD8⁺T cells are from tumors.

In some embodiments of the methods of the invention, Treg function is inhibited as compared to prior to administration of the combination. In some embodiments, T cell fatigue is reduced as compared to prior to administration of the combination.

In some embodiments, the number of tregs is reduced as compared to before administration of the combination. In some embodiments, the level of plasma IFN- γ is increased as compared to prior to administration of the combination. Can be measured, for example, by measuring CD4⁺Fox3p⁺CD45⁺The percentage of cells (e.g., by FACS analysis) to assess Treg number. In some embodiments, the absolute number of tregs in, for example, a sample is determined. In some embodiments, the tregs are from peripheral blood. In some embodiments, the tregs are from a tumor.

In some embodiments, priming, activation, and/or proliferation of T cells is increased as compared to prior to administration of the combination. In some embodiments, the T cell is CD4⁺And/or CD8⁺T cells. In some embodiments, by determining Ki67⁺CD8⁺T cell proliferation is detected by percentage of T cells (e.g., by FACS analysis). In some embodiments, by determining Ki67⁺CD4⁺T cell proliferation is detected by percentage of T cells (e.g., by FACS analysis). In some embodiments, the T cells are from peripheral blood. In some embodiments, the T cell is from a tumor.

5. Detection and diagnostic method

According to the present invention, nuclear PKC-theta and ZEB1 are useful as biomarkers of T-cell mesenchymal phenotype and impaired T-cell function. In addition, as is known in the art, PD-1, TBET and EOMES can be used to assess T cell exhaustion. T cells can be obtained from a patient sample containing T cells, which is a suitably selected tissue sample, such as a tumor, and a liquid sample, such as peripheral blood. In some embodiments, the sample is obtained prior to treatment with the combination therapy. In some embodiments, the tissue sample is a formalin-fixed and paraffin-embedded, archive-retained, fresh or frozen sample. In some embodiments, the sample is whole blood. In some embodiments, the whole blood comprises immune cells, circulating tumor cells, and any combination thereof.

The presence and/or expression level/amount of biomarkers (e.g., any one or more of PKC-theta, ZEB1, TBET, and EOMES, also collectively referred to herein as "T cell function biomarkers") including, but not limited to, DNA, mRNA, cDNA, proteins, protein fragments, and/or gene copy number can be determined qualitatively and/or quantitatively based on any suitable criteria known in the art. In certain embodiments, the presence and/or expression level/amount of the biomarker in the first sample is increased or elevated as compared to the presence/absence and/or expression level/amount in the second sample (e.g., prior to treatment with the combination therapy). In certain embodiments, the presence/absence and/or expression level/amount of the biomarker in the first sample is reduced or decreased as compared to the presence and/or expression level/amount in the second sample. In certain embodiments, the second sample is a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. Additional disclosure for determining the presence/absence and/or expression level/amount of a gene is described herein.

In some embodiments of any of the methods, elevated expression refers to an overall increase in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain embodiments, elevated expression refers to an increase in the expression level/amount of a biomarker in a sample, wherein the increase is at least about any of 1.5x, 1.75x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 25x, 50x, 75x, or 100x of the expression level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression refers to an overall increase of greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, internal control (e.g., housekeeping gene).

In some embodiments of any of the methods, reduced expression refers to an overall reduction in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain embodiments, reduced expression refers to a reduction in the expression level/amount of a biomarker in a sample, wherein the reduction is at least about any of 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1x, 0.05x, or 0.01x of the expression level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

The presence and/or expression levels/amounts of various biomarkers in a sample can be analyzed by a variety of methods, many of which are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemistry ("IHC"), western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR"), including quantitative real-time PCR ("qRT-PCR") and other amplification type detection methods, such as branched DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling, and/or serial analysis of gene expression ("SAGE"), PCR, And any of a variety of assays that can be performed by protein, gene, and/or tissue array analysis. Typical Protocols for assessing the status of genes and gene products can be found, for example, In Ausubel et al, eds. 1995, Current Protocols In molecular biology, units 2(Northern Blotting), 4(Southern Blotting), 15 (immunoblotting) and 18(PCR analysis). Multiplex immunoassays may also be used, such as those available from Rules Based Medicine or Meso scale discovery ("MSD").

In some embodiments, the presence and/or expression level/amount of a biomarker is determined using a method comprising: (a) performing gene expression profiling, PCR (e.g., rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH on a sample (e.g., a cancer sample); and b) determining the presence and/or expression level/amount of the biomarker in the sample. In some embodiments, microarray methods include the use of microarray chips having one or more nucleic acid molecules that can hybridize under stringent conditions to nucleic acid molecules encoding the above-described genes, or having one or more polypeptides (e.g., peptides or antibodies) that can bind to one or more proteins encoded by the above-described genes. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.

Methods for assessing mRNA in a cell are well known and include, for example, hybridization assays using complementary DNA probes (e.g., in situ hybridization using labeled ribonucleic acid probes (riboprobes) specific for one or more genes, Northern blots, and related techniques) and various nucleic acid amplification assays (e.g., RT-PCR using complementary primers specific for one or more genes, as well as other amplification type detection methods, such as branched DNA, SISBA, TMA, and the like).

The mRNA of a mammalian sample can be conveniently determined using Northern, dot blot or PCR analysis. In addition, such methods can include one or more steps that allow for the determination of target mRNA levels in a biological sample (e.g., by simultaneously examining the levels of comparative control mRNA sequences for "housekeeping" genes (e.g., actin family members)). Optionally, the sequence of the amplified target cDNA can be determined.

Alternative methods include protocols for examining or detecting mRNA (e.g., target mRNA) in a tissue or cell sample by microarray technology. Test and control mRNA samples from the test and control tissue samples were reverse transcribed and labeled using a nucleic acid microarray to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured so that the sequence and location of each member in the array is known. For example, selected genes whose expression correlates with increased or decreased clinical benefit of anti-angiogenic therapy can be arrayed on a solid support. Hybridization of a labeled probe to a particular array member indicates that the sample from which the probe is derived expresses the gene.

According to some embodiments, the presence and/or expression level/amount is measured by observing the protein expression level of the aforementioned genes. In certain embodiments, the methods comprise contacting the biological sample with an antibody to a biomarker described herein (e.g., an anti-PD-1 antibody, an anti-PKC-theta antibody, an anti-TBET antibody, an anti-ZEB antibody, an anti-EOMES antibody) under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods may be in vitro or in vivo. In some embodiments, one or more anti-biomarker antibodies are used to select a subject suitable for combination therapy with a PKC-theta inhibitor and a PD-1 binding antagonist.

In certain embodiments, the presence and/or expression level/amount of a biomarker protein in a sample is examined using IHC and staining protocols. IHC staining of tissue sections has proven to be a reliable method for determining or detecting the presence of proteins in a sample. In some embodiments, the expression of a T cell function biomarker in a sample from the individual is elevated protein expression, and in further embodiments, an IHC assay is used. In one embodiment, the expression level of a biomarker is determined using a method comprising: (a) IHC analysis of a sample (e.g., a subject cancer sample) with an antibody; and b) determining the expression level of the biomarker in the sample. In some embodiments, IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line stained sample or tissue sample from a non-cancer patient).

In some embodiments, T cell function biomarker expression is assessed on a tumor or tumor sample. As used herein, a tumor or tumor sample may encompass part or all of the tumor area occupied by tumor cells. In some embodiments, the tumor or tumor sample can further encompass the region of the tumor occupied by tumor-associated intratumoral cells and/or tumor-associated stroma (e.g., connective tissue proliferative (desmoplastic) stroma around a continuous tumor). The tumor-associated intratumoral cells and/or tumor-associated matrix can include an area of immune infiltration (e.g., tumor-infiltrating immune cells described herein) immediately adjacent to and/or contiguous with the main tumor mass. In some embodiments, expression of a T cell functional biomarker is assessed on tumor cells. In some embodiments, expression of a T cell functional biomarker is assessed on immune cells within a tumor region (e.g., tumor-infiltrating immune cells) as described above.

In an alternative method, the sample may be contacted with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex, and the complex then detected. The presence of biomarkers can be detected in a variety of ways, for example, by analyzing a variety of tissues and samples, including plasma or serum, by western blot and ELISA methods. A variety of immunoassay techniques are available using this assay format, see, e.g., U.S. Pat. nos. 4,016,043, 4,424,279 and 4,018,653. These include both single site and two site or "sandwich" assays of the non-competitive type, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target biomarkers.

The presence and/or expression level/amount of a selected T cell functional biomarker in a tissue or cell sample can also be examined by function-based or activity-based assays. For example, if the biomarker is an enzyme (e.g., PKC-theta), an assay known in the art (e.g., a kinase assay) may be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.

In certain embodiments, the samples are normalized for differences in the amount of biomarker determined and differences in the mass of the sample used and differences between assay runs. Such normalization can be achieved by detecting and incorporating the expression of certain normalized biomarkers, including well-known housekeeping genes. Alternatively, normalization can be based on the mean or median signal of all genes assayed or a larger subset thereof (global normalization approach). The normalized amount of the target tumor mRNA or protein measured is compared to the amount found in the reference group on a gene-by-gene basis. The normalized expression level of each mRNA or protein in each test tumor of each subject can be expressed as a percentage of the expression level measured in the reference group. The presence and/or expression level/amount measured in a particular subject sample to be analyzed will fall within certain percentiles of this range, which can be determined by methods well known in the art.

In some embodiments, the sample is a clinical sample. In other embodiments, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsies are commonly used to obtain representative pieces of tumor tissue. Alternatively, the tumor cells may be obtained indirectly in the form of tissues or body fluids known or believed to contain the tumor cells of interest. For example, samples of lung cancer lesions may be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushing, or from sputum, pleural fluid, or blood. Genes or gene products can be detected from cancer or tumor tissue or other body samples (e.g., urine, sputum, serum, or plasma). The same techniques discussed above for detecting target genes or gene products in cancerous samples can be applied to other body samples. Cancer cells may be shed from cancerous lesions and appear in such body samples. By screening these body samples, a simple early diagnosis of these cancers can be made. In addition, by testing for target genes or gene products in these body samples, the progress of the treatment can be more easily monitored.

In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined plurality of samples from the same subject or individual obtained at one or more different time points than the test sample obtained. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at an earlier time point than the test sample. Such a reference sample, reference cell, reference tissue, control sample, control cell or control tissue may be useful if the reference sample is obtained during the initial diagnosis of cancer and the test sample is subsequently obtained at the time of cancer metastasis.

In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals that are not the subject or the individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals who are not subjects or individuals who have a disease or disorder (e.g., cancer). In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from normal tissue or pooled plasma or serum samples from one or more individuals other than the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissue or pooled plasma or serum samples from one or more individuals who are not the subject or the individual having a disease or condition (e.g., cancer).

In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a tumor tissue sample (e.g., biopsy). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is kidney tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is stomach tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, and/or bone/bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is endometrial tissue. In some embodiments, the tissue sample is brain tissue (e.g., glioblastoma, neuroblastoma, etc.).

In some embodiments, a tumor tissue sample (the terms "tumor sample" are used interchangeably herein) may encompass part or all of the tumor area occupied by tumor cells. In some embodiments, the tumor or tumor sample can further encompass the region of the tumor occupied by tumor-associated intratumoral cells and/or tumor-associated stroma (e.g., connective tissue proliferative (desmoplastic) stroma around a continuous tumor). The tumor-associated intratumoral cells and/or tumor-associated matrix can include an immune-infiltrated area (e.g., tumor-infiltrated immune cells as described herein) immediately adjacent and/or contiguous to the main tumor mass.

In some embodiments, tumor cell staining is expressed as a percentage of all tumor cells that exhibit membrane staining of any intensity. The infiltrated immune cell staining can be expressed as a percentage of the total tumor area occupied by immune cells showing any intensity staining. The total tumor area includes malignant cells and tumor-associated stroma, including the immediately adjacent and immunoinfiltrated areas adjacent to the main tumor mass. In addition, the staining of infiltrating immune cells can be expressed as a percentage of all tumor infiltrating immune cells.

In some embodiments of any of the methods, the disease or disorder is a tumor. In some embodiments, the tumor is a malignant cancerous tumor (i.e., cancer). In some embodiments, the tumor and/or cancer is a solid tumor or a non-solid or soft tissue tumor. Examples of soft tissue tumors include leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphocytic leukemia, acute myelogenous leukemia, mature B-cell acute lymphocytic leukemia, chronic lymphocytic leukemia, prolymphocytic leukemia, or hairy cell leukemia) or lymphoma (e.g., non-hodgkin's lymphoma, cutaneous T-cell lymphoma, or hodgkin's disease). Solid tumors include cancers of any body tissue other than the blood, bone marrow, or lymphatic system. Solid tumors can be further divided into tumors of epithelial and non-epithelial origin. Examples of epithelial solid tumors include tumors of the gastrointestinal tract, colon, colorectal (e.g., basal colorectal cancer), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., endometrioid ovary), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, labia, nasopharynx, skin, uterus, male reproductive organs, urinary organs (e.g., urothelium, hyperplastic urothelium, transitional cell carcinoma), bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors and bone tumor tumors. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is second or third line locally advanced or metastatic non-small cell lung cancer. In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor at a second site, which is derived from any of the types of cancer described above.

In some embodiments of any of the methods, the cancer exhibits (e.g., is infiltrated by) human effector cells. Methods for detecting human effector cells are well known in the art and include, for example, by IHC. In some embodiments, the cancer exhibits high levels of human effector cells. In some embodiments, the human effector cell is one or more of an NK cell, a macrophage, a monocyte. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

In some embodiments of any of the methods, the cancer exhibits FcR expressing cells (e.g., is infiltrated by FcR expressing cells). Methods for detecting FcR are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of FcR expressing cells. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activated Fc γ R. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

In some embodiments, the T cell function biomarker is detected in the sample using a method selected from the group consisting of: FACS, western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH and combinations thereof. In some embodiments, the T cell functional biomarkers are detected using FACS analysis. In some embodiments, the biomarker of T cell function is PD-1. In some embodiments, PD-1 expression is detected in a blood sample. In some embodiments, PD-1 expression is detected on circulating immune cells in a blood sample. In some embodiments, the circulating immune cell is CD3⁺/CD8⁺T cells. In some embodiments, the immune cells are isolated from the blood sample prior to analysis. Any suitable method may be used to isolate/enrich for such cell populations, including but not limited to cell sorting. In some embodiments, PD-1 expression is decreased in a sample from an individual that is responsive to treatment with a PKC-theta inhibitor and/or a PD-1 binding antagonist (e.g., an anti-PD-1 antibody). In some embodiments, PD-1 expresses circulating immune cells (e.g., CD 3) in a blood sample⁺/CD8⁺T cells).

Also provided herein are diagnostic methods and kits based on determining the co-localization of PKC-theta and ZEB1 in the nucleus, and that this co-localization contributes at least in part to the suppression of the EMT of T cells and their immune function. The diagnostic method suitably comprises: (i) obtaining a sample from a subject, wherein the sample comprises T cells (e.g., CD 8)⁺T cells); (ii) bringing the sample into contact withContacting a first binding agent that binds to PKC-theta in the sample with a second binding agent that binds to ZEB1 in the sample; and (iii) detecting the localization of the first and second binding agents in the nucleus of the T cell, wherein localization of the first and second binding agents in the nucleus of the T cell indicates the presence of a T cell dysfunctional disorder in the subject.

The first and second binding agents suitably bind to an epitope of PKC-theta and ZEB1 polypeptides, respectively. Any suitable epitope may be selected in the amino acid sequence of PKC-theta (e.g., as shown in GenPept accession nos. XP _005252553, XP _005252554, XP _005252555, and XP _ 005252556), or in the amino acid sequence of ZEB1 (e.g., as shown in GenPept accession nos. NP _00131058, NP _001310579, NP _001310586, and NP _ 001310601).

The localization of PKC-theta and ZEB1 in the nucleus of T cells can be performed using any suitable localization technique, such as by IHC, which typically uses an anti-PKC-theta antibody having a different detectable moiety or label than the anti-ZEB 1 antibody. In some embodiments, a spatial proximity assay (also referred to as a "proximity assay") is employed that can be used to assess complex formation between PKC-theta and ZEB 1. Proximity assays rely on the principle of "proximity detection", in which an analyte (typically an antigen) is detected by the simultaneous binding of multiple (i.e. two or more, typically two, three or four) binding reagents or probes that are in proximity by binding to the analyte (hence the term "proximity probes"), thereby allowing a signal to be generated.

In some embodiments, at least one proximity probe comprises a nucleic acid domain (or portion) linked to the analyte binding domain (or portion) of the probe, and generation of a signal comprises interaction of the nucleic acid portion and/or other functional portions carried by other probes. Thus, the generation of a signal is dependent on the interaction between the probes (more specifically, on the interaction of the nucleic acids or other functional moieties/domains carried by the probes), and therefore the signal is generated only when both (or more) of the necessary probes have bound to the analyte, thereby improving specificity of the detection system. In recent years, the concept of proximity detection has been developed, and many assays based on this principle are now well known in the art.

Proximity assays are typically used to assess the close proximity of two specific proteins or portions thereof, e.g., proteins that bind to each other, fusion proteins, and/or closely adjacent proteins. One such assay used in some embodiments of the invention is called the Proximity Ligation Assay (PLA), which is characterized by two antibodies (produced in different species) that bind to a target of interest (see Nature Methods 3,995-1000 (2006)). PLA probes (species-specific secondary antibodies with attached unique oligonucleotide strands) are then conjugated with appropriate primary antibodies. In the case of very close proximity of the target, the oligonucleotide strand of the PLA probe can interact with other ssDNA and DNA ligases, and can therefore be circularized and amplified by Rolling Circle Amplification (RCA). When highly processive DNA polymerases such as Phi29DNA polymerase are used, circular DNA templates can replicate hundreds to thousands of times, thereby producing ssDNA molecules ranging from hundreds of nanometers to micrometers in length (see angelwaldte chemical International Edition,2008,47, 6330-. After amplification, the replicated DNA may be detected by a detection system. Thus, the visible signal indicates that the target of interest is in close proximity. These assays are characterized by the use of several DNA-antibody conjugates as well as enzymes such as DNA ligase and DNA polymerase.

In other embodiments, a dual binding reagent (DB) assay is employed that utilizes a bispecific detection agent consisting of two Fab fragments with rapid dissociation kinetics linked by a flexible linker (Van diack et al, 2014Chemistry & Biology Vol.21 (3): 357-368). In principle, since the dual binding agent comprises Fab fragments with fast dissociation kinetics, the dual binding agent will be washed away if only one Fab fragment binds to its epitope (simultaneous co-binding of the two Fab fragments of the dual binding agent will prevent dissociation of the dual binding agent and lead to positive staining/visibility).

According to another method disclosed in international publication WO2014/139980, which is included in the practice of the present invention, proximity assays and tools are described that utilize biotin ligase substrates and enzymes for the proximity assay. The method provides for the detection of target molecules and proximity while maintaining the cellular environment of the sample. The use of biotin ligase (e.g., an enzyme from E.coli) and a peptide substrate (e.g., an amino acid substrate for the enzyme) provides a sensitive and specific detection of protein-protein interactions in FFPE samples. Because biotin ligase can effectively biotinylate the correct peptide substrate in the presence of biotin and only react when the enzyme is in physical contact with the peptide substrate, the biotin ligase and substrate can be conjugated separately to two antibodies that each recognize the target of interest.

Also provided herein are methods of monitoring pharmacodynamic activity of a PD-1 binding antagonist treatment, by measuring the expression level of one or more T cell functional biomarkers as described herein in a sample comprising leukocytes obtained from a subject, wherein the subject has been treated with a PD-1 binding antagonist and a PKC-theta inhibitor, and wherein the one or more T cell function biomarkers are selected from the group consisting of nuclear PKC-theta, ZEB1, TBET, PD-1, and EOMES, determining the pharmacodynamic activity exhibited by the treatment based on the expression level of one or more T cell function biomarkers in a sample obtained from the subject compared to a reference, a pharmacodynamic activity of a PD-1 antagonist treatment is indicated when the expression level of one or more T cell functional biomarkers is increased as compared to a reference. These methods may further comprise measuring one or more T cell functions and/or cellular composition (e.g., percentage of tregs and/or absolute number of tregs; e.g., CD8⁺Number of effector T cells), wherein the other biomarker of T cell function comprises a cytokine, such as IFN- γ, a T cell marker, or a memory T cell marker (e.g., a marker of T effector memory cells); determining the pharmacodynamic activity exhibited by the treatment based on the expression level of one or more T cell function biomarkers, one or more other T cell function biomarkers, and/or cellular composition in the sample obtained from the subject compared to a reference, the one or more T cell function biomarkers when compared to the referenceAn increase in the expression level of a marker, one or more other T cell function biomarkers, and/or cellular composition is indicative of pharmacodynamic activity of the PD-1 antagonist treatment. The expression level of a biomarker and/or cellular composition may be measured by one or more of the methods described herein.

As used herein, "Pharmacodynamic (PD) activity" may refer to a therapeutic (e.g., PKC-theta inhibitor in combination with PD-1 binding antagonist therapy) effect on a subject. Examples of PD activity may include modulating the expression level of one or more genes. Without wishing to be bound by theory, it is believed that monitoring PD activity (e.g., by measuring expression of one or more T cell functional biomarkers) may be advantageous during clinical trials to detect PKC-theta inhibitors and PD-1 binding antagonists. Monitoring PD activity can be used, for example, to monitor response to therapy, toxicity, and the like.

In some embodiments, the expression level of one or more marker genes, proteins, and/or cellular constituents may be compared to a reference, which may include a sample from a subject not receiving treatment (e.g., treatment with a PKC-theta inhibitor in combination with a PD-1 binding antagonist). In some embodiments, a reference may comprise a sample from the same subject prior to receiving treatment (e.g., treatment with a PKC-theta inhibitor in combination with a PD-1 binding antagonist). In some embodiments, a reference may comprise reference values from one or more samples from other subjects receiving treatment (e.g., treatment with a PKC-theta inhibitor in combination with a PD-1 binding antagonist). For example, a population of patients may be treated, and an average, mean or median value of one or more gene expression levels may be generated from the population as a whole. A set of samples obtained from cancers having common characteristics (e.g., the same cancer type and/or stage, or exposure to common treatments, such as PKC-theta inhibitor treatment in combination with a PD-1 binding antagonist) may be studied, e.g., for clinical outcome studies, from a population. The panel can be used to derive a reference, e.g., a reference number, for comparison to a sample of the subject. Any reference described herein can be used as a reference to monitor PD activity.

Certain aspects of the present disclosure relate to measuring the expression level of one or more biomarkers (e.g., gene expression products including mRNA and protein) in a sample. In some embodiments, the sample may comprise leukocytes. In some embodiments, the sample can be a peripheral blood sample (e.g., from a patient having a tumor). In some embodiments, the sample is a tumor sample. Tumor samples may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell type associated with a tumor. In some embodiments, the sample is a tumor tissue sample containing tumor infiltrating leukocytes. In some embodiments, a sample can be treated to separate or isolate one or more cell types (e.g., leukocytes). In some embodiments, the sample can be used without separating or isolating the cell types.

Tumor samples may be obtained from a subject by any method known in the art, including but not limited to biopsy, endoscopy, or surgery. In some embodiments, a tumor sample can be prepared by: such as freezing, fixing (e.g., by using formalin or similar fixative), and/or embedding in paraffin. In some embodiments, a tumor sample can be sectioned. In some embodiments, a fresh tumor sample (i.e., a sample that has not been prepared by the methods described above) may be used. In some embodiments, tumor samples may be prepared by incubation in solution to preserve the integrity of mRNA and/or protein.

In some embodiments, the sample may be a peripheral blood sample. Peripheral blood samples may include leukocytes, PBMCs, and the like. Any technique known in the art for isolating leukocytes from a peripheral blood sample can be used. For example, a blood sample may be drawn, red blood cells may be lysed, and a white blood cell pellet may be isolated and used for the sample. In another example, density gradient separation can be used to separate leukocytes (e.g., PBMCs) from erythrocytes. In some embodiments, a fresh peripheral blood sample (i.e., a sample that has not been prepared by the methods described above) may be used. In some embodiments, peripheral blood samples may be prepared by incubation in solution to preserve mRNA and/or protein integrity.

In some embodiments, the response to treatment may refer to any one or more of: extending survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating the signs or symptoms of cancer. In some embodiments, according to a set of RECIST guidelines disclosed for determining tumor status (i.e., response, stability, or progression) in a cancer patient, a response may refer to an improvement in one or more factors. For a more detailed discussion of these guidelines, see Eisenhauer et al (2009Eur J Cancer 45: 228-47), Topalian et al (2012N Engl JMed 366: 2443-54), Wolchok et al (2009Clin Can Res 15: 7412-20) and Therasse et al (2000J. Natl. Cancer Inst.92: 205-16). A responsive subject can refer to a subject whose cancer shows improvement, e.g., based on one or more factors based on RECIST criteria. A non-responsive subject may refer to a subject whose cancer does not show improvement, e.g., based on one or more factors based on RECIST criteria.

Conventional response criteria may not be sufficient to characterize the anti-tumor activity of the therapeutic agents of the present invention, which may produce a delayed response, including the appearance of new lesions, that may occur before the initial apparent radiological progression. Accordingly, revised response criteria have been developed that take into account the potential for new lesions to appear and allow confirmation of radiologic progression in subsequent assessments. Thus, in some embodiments, a response may refer to an improvement in one or more factors according to an immune-related response criteria (irRC). See, e.g., Wolchok et al (2009, supra). In some embodiments, new lesions are added to the determined tumor burden and then used for radiologic progression, for example in subsequent assessments. In some embodiments, the presence of a non-target lesion is included in the assessment of complete response, not in the assessment of radiologic progression. In some embodiments, radiologic progression may be determined based solely on measurable disease and/or may be confirmed by a continuous assessment of ≧ 4 weeks from the date of first recording.

In some embodiments, the response may comprise immune activation. In some embodiments, the response may include a therapeutic efficacy. In some embodiments, the response may include immune activation and therapeutic efficacy.

6. Reagent kit

In other aspects of the invention, therapeutic kits are provided comprising a PKC-theta inhibitor and a PD-1 binding antagonist. In some embodiments, the therapeutic kit further comprises a package insert comprising instructional material for the simultaneous administration of the PKC-theta inhibitor and the PD-1 binding antagonist for the treatment of a T cell dysfunctional disorder or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, or for treating or delaying progression of cancer, or for treating an infection in an individual. Any PKC-theta inhibitor described herein or known in the art and PD-1 binding antagonist may be included in the kit.

In some embodiments, the PKC-theta inhibitor and the PD-1 binding antagonist are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or hastelloy). In some embodiments, the container contains the formulation, and a label on or associated with the container can indicate the instructions for use. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructional materials. In some embodiments, the kit further comprises one or more additional agents (e.g., chemotherapeutic agents and antineoplastic agents). Suitable containers for one or more reagents include, for example, bottles, vials, bags, and syringes.

In other embodiments of the present invention, diagnostic kits for determining the expression of biomarkers, including the T cell functional biomarkers disclosed herein, are provided, comprising reagents that allow for the detection and/or quantification of the biomarkers. Such reagents include, for example, a compound or material, or a group of compounds or materials, which allows for quantification of a biomarker. In particular embodiments, the compound, material or group of compounds or materials allows for the determination of the expression level of a gene (e.g., a T cell functional biomarker gene), including but not limited to the extraction of RNA material, the determination of the level of the corresponding RNA, etc., primers used to synthesize the corresponding cDNA, primers used to amplify DNA, and/or probes capable of specifically hybridizing to the RNA encoded by the gene (or the corresponding cDNA), TaqMan probes, proximity assay probes, ligases, antibodies, etc.

The kit may also optionally include appropriate reagents for detecting the marker, positive and negative controls, wash solutions, blotting membranes, microtiter plates, dilution buffers, and the like. For example, a nucleic acid-based detection kit can include (i) a T cell functional biomarker polynucleotide (which can serve as a positive control), (ii) a primer or probe that specifically hybridizes to the T cell functional biomarker polynucleotide. Enzymes suitable for amplifying nucleic acids, including various polymerases (reverse transcriptase, Taq, SequenaseTM, DNA ligase, etc., depending on the nucleic acid amplification technique used), deoxynucleotides and buffers may also be included to provide the reaction mixture required for amplification. Such kits will also typically contain, in a suitable manner, a different container for each individual reagent and enzyme, as well as for each primer or probe. Alternatively, the protein-based detection kit can include (i) a T cell function biomarker polypeptide (which can serve as a positive control), (ii) an antibody that specifically binds to the T cell function biomarker polypeptide. The kit also features various (e.g., one or more) devices and (e.g., one or more) reagents for performing an assay described herein; and/or printed instructional material for quantifying expression of a T cell functional biomarker gene using the kit. The reagents described herein, which optionally may be associated with a detectable label, may be present in the form of a microfluidic card, chip or chamber, microarray or kit, suitable for use in the assays described in the examples or below, e.g., the RT-PCR or Q-PCR techniques described herein.

Materials suitable for packaging the components of the diagnostic kit may include crystals, plastics (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, envelopes, and the like. In addition, the kits of the invention may comprise instructional materials for the simultaneous, sequential or separate use of the different components comprised in the kit. The instructional material may be in the form of printed material or in the form of an electronic support capable of storing instructions, such as an electronic storage medium (disk, tape, etc.), an optical medium (CD-ROM, DVD), etc., for the subject to read the instructions. Alternatively or additionally, the medium may contain an internet address that provides instructional material.

In order that the invention may be readily understood and put into practical effect, specific preferred embodiments will now be described by way of the following non-limiting examples.

Examples

Example 1

PKC-theta as a target for therapeutic intervention

The present inventors have developed novel peptide inhibitors specific in inhibiting PKC-theta entry into the nucleus, which are disclosed in PCT/AU2017/050083 filed on 1/2/2017. One of the inhibitors of these peptides, RKEIDPPFRPKVK (also referred to herein as "PKC θ i"), was tested in MCF7 breast cancer cell line to determine its effect on multiple PKC isoforms (β 2, β 1, α, and γ) and PKC- θ, as shown in fig. 1A, B and C for structural and physical properties. The PKC θ i peptide has been shown to significantly inhibit nuclear localization of PKC- θ, while having no effect on other PKC isoforms, demonstrating its target specificity (fig. 1D). The peptide inhibitor also significantly inhibited proliferation of MCF7 cells (FIG. 1E), without affecting the catalytic activity of PKC-theta, suggesting that it acts in a manner that inhibited the nuclear axis of PKC-theta (FIG. 1F).

Method of producing a composite material

Cell treatment:

treated cells were permeabilized by incubation with 1% Triton X-100 for 20 minutes and probed with rabbit primary antibodies directed against PKC-theta (T538p), PKC-beta 1, PKC-beta 2, PKC-alpha, PKC-and PKC-gamma and visualized with donkey anti-rabbit AF 488. Coverslips were mounted on glass microscope slides using ProLong Diamond anti-fade reagent (Life Technologies). And (3) positioning the protein target by a confocal laser scanning microscope. Single 0.5 μm sections were obtained using a Leica DMI8 microscope (run the LAX software using 100x oil immersion lens). The final image is obtained by averaging four successive images of the same slice. The digital images were analyzed using ImageJ software (ImageJ, NIH, besiesda, maryland, usa) to determine Total Nuclear Fluorescence Intensity (TNFI), Total Cytoplasmic Fluorescence Intensity (TCFI), or Total Fluorescence Intensity (TFI). The fluorescence ratio of nucleus to cytoplasm (Fn/c) was calculated using the following formula: fn/c ═ (Fn-Fb)/(Fc-Fb), where Fn is nuclear fluorescence, Fc is cytoplasmic fluorescence, and Fb is background fluorescence, were used to determine the effect on nuclear localization.

Mouse model MDA-MB-231 mouse xenografts:

five-week old female nude mice were obtained from the animal resource center (Perth) and acclimatized in the animal facility of the John Keting medical college (JCSMR) for one week prior to the experiment all experimental procedures had been evaluated and approved by the animal Experimental ethics Committee of the Australian national university (ethical code A2014/30). MDA-MB-231 human breast cancer cells were injected subcutaneously into the right breast (2 × 10)⁶Cells, in a 1: 1PBS and BD Matrigel Matrix). Tumors were measured using external calipers and calculated using the modified ellipse equation: 1/2(a/b2), where a is the longest diameter and b is the shortest diameter. Tumors were allowed to grow to approximately 50mm before starting treatment³(about 15 days). All treatments were given by IP injection with 40mg/kg of PKC nuclear inhibitor. Tumors were excised and collected in DMEM supplemented with 2.5% FCS. The tumors were then minced using a scalpel and incubated in DMEM 2.5% FCS and collagenase type 4 (Worthington-Biochem) (1mg collagenase per 1g of tumor) for 1 hour at 37 ℃. Digested tumors were spun and resuspended in DMEM in 2.5% FCS, then processed through 0.2 μ M filters and analyzed on Nanostring platform.

Example 2

BRAF-negative melanoma patient CD8⁺Expression signature of PKC-theta in T cells

The inventors examined CD8 in patients with BRAF-negative melanoma who received PD-1 immunotherapy⁺Expression of PKC-theta in T cells to determine whether PKC-theta has a role in tolerance to treatment with such immune checkpoint inhibitors. To is coming toCharacterisation of CD8⁺PKC-theta profiles in T cells examined the expression of this biomarker in FFPE tissue from BRAF negative melanoma tissue biopsies of melanoma patients divided into 4 groups according to RECIST 1.1 response to immunotherapy, as summarized in table 1.

TABLE 1

Examination of FFPE tissue from BRAF-negative melanoma tissue biopsy for CD8⁺The expression of PKC-theta and PD1 in T cells found that PD1 was expressed only in the Complete Response (CR) and Stable Disease (SD) groups, whereas PKC-theta was highly expressed in the PD group (FIG. 2A). IL-2, IFN-gamma and TNF- α were markers of T cell activation and effector capacity, while ZEB1 was a negative regulator of T cell response, associated with inhibition of T cell effects and progression of cancer.

Next, the expression of ZEB1 repressor protein and PKC-theta was analyzed in FFPE samples from melanoma patients, and the results presented in figure 2C indicate that only the PD group had significant expression of ZEB1, and in this group, the patient samples with dual immunotherapy tolerance had the highest ZEB1 expression.

These data indicate that in patients with tolerance to immunotherapy, CD8 is present⁺In T cells, expression of IL-2, IFN- γ and TNF- α was low, and ZEB1 was up-regulated.

Next, the present inventors examined CD8 isolated from blood of melanoma patients⁺Expression signature of PKC- θ in T cells (FIG. 3). Treatment of CD8 with vehicle control or PMA/CI⁺T cells, then treated with a mimetic or PKC θ i. Then use the point toCD8 (targeting CD 8)⁺T cells), ZEB1, and PKC-theta antibodies probe cells. Interestingly, the expression of ZEB1 and PKC-theta was found to be highest in PD (patients with primary tolerance to both single and double immunotherapy) groups, while the expression of ZEB1 and PKC-theta was lower in patients in response to SD and CR groups. Notably, PD/PR/PD group samples that were converted to PR group and then relapsed had moderate to high expression of ZEB1 and PKC-theta between SD and PD groups (fig. 3A). Pearson Correlation Coefficient (PCC) of PKC-theta and ZEB1 was also evaluated to judge the extent of co-localization, and the data strongly suggests that these two proteins are significantly co-localized in the nuclei of melanoma patients, highest with PCC in the PD group. Treatment with PKC θ i peptide inhibitors significantly abolished nuclear PKC- θ and ZEB1 expression in all patient samples.

Next, CD8 processed as above⁺Markers of T cell activity were examined in T cells, IFN- γ and TNF- α (fig. 3B) the inventors found, consistent with the data of fig. 2A, that the PD group had the lowest expression of IFN- γ and TNF- α, while the CR/SD group (responsive to immunotherapy) and the PD/PR/PD group had higher levels of IFN- γ and TNF- α.

This data set clearly demonstrates the powerful role that PKC-theta overexpression plays in inhibiting T-cell based immune responses in metastatic melanoma patients, and one of the major mechanisms mediating this inhibition is the repressor ZEB1, inhibition of the nuclear axis of PKC-theta and subsequent ZEB1, rescuing expression of the T-cell activation markers IFN- γ and TNF- α, which again indicates that PKC θ i peptide inhibitors have a powerful potential to directly target Cancer Stem Cells (CSCs) and Circulating Tumor Cells (CTCs), and at the same time, to rescue CD8⁺T cell mediated immune responses, which may improve outcome in metastatic cancer patients, including patients with advanced metastatic melanoma.

Method of producing a composite material

Patient categories:

melanoma patients were selected for this biomarker study, using RECIST 1.1 analysis as described, patients were divided into 4 groups (single or dual therapy with pembrolizumab, nivolumab, and/or leprimumab) based on response to immunotherapy, into the following treatment response groups based on assessment of target lesions: complete Response (CR): all target lesions disappeared. The minor axis of any pathological lymph node (whether targeted or non-targeted) must be reduced to <10 mm; partial Response (PR): the sum of target lesion diameters is reduced by at least 30% relative to the sum of baseline diameters. Stable Disease (SD): neither is there a sufficient reduction to be PR nor a sufficient increase to be PD. Progressive Disease (PD): the sum of the target lesion diameters increased by at least 20%, and the absolute increase was at least 5mm (the appearance of one or more new lesions was also considered to be progression). Samples were taken every 3 months for 24 months after baseline blood sampling.

Cell preparation and treatment:

using RosetteSep^TMMethod for pre-enriching metastatic melanoma biopsy samples using RosetteSep^TMHuman CD45 depletion kit (15162, Stemcell Technologies) for CTC isolation using SepMate^TM-50(IVD) density gradient tubes (85450, Stemcell Technologies) and Lymphoprep^TMDensity gradient media (07861, stemcell technologies) were subjected to density gradient centrifugation to remove CD45+ cells and red blood cells, then enriched CTC cells were preclinically screened with 8 μm concentrations of control or PKC θ i peptide inhibitors, then enriched cells were centrifuged, smeared onto coverslips pretreated with poly-1-lysine and fixed, then stored in PBS for staining, CTCs were incubated with 1% Triton X-100 for permeabilization and probed with rabbit anti-PKC- θ (T538p), mouse anti-CSV and goat anti-ABCB 5, and probed with donkey anti-rabbit AF488, anti-mouse 568 and anti-goat 633 for visualization, for examination of PKC CD- θ 8T cells treated with PKC θ i (T538), goat anti-rabbit anti-TNF 635, mouse anti-TNF 38735, gamma-IFN α, goat anti-mouse anti-TNF-goat anti-goat 63633, goat anti-TNF-goat anti-goat 635, goat anti-TNF-goat anti-goat 63633, visualized with rabbit anti-TNF-5, goat anti-1, anti-goat 633874, and goat anti-goatVisualization was performed in mouse 568 and anti-goat 633. Coverslips were mounted on glass microscope slides using ProLongDiamond antibody reagent (Life Technologies). The protein target was localized by confocal laser scanning microscopy. Single 0.5 μm sections were obtained using a Leica DMI8 microscope (run the LAX software using 100x oil immersion lens). The final image is obtained by averaging four successive images of the same slice. The digital images were analyzed using ImageJ software (ImageJ, NIH, besistar, maryland, usa) to determine TNFI, TCFI or TFI. Fluorescence signal intensity was plotted along a single line across nuclei using the plotted profile feature of ImageJ (n ═ 5 lines per nucleus, 5 individual cells), for each on-line point, using the mean fluorescence signal intensity of the indicated antibody pairs plotted, with SE. Signal plots were drawn to compare the change in signal for each antibody compared to the opposite antibody. For each plot profile, the PCC was determined. PCC represents the intensity of the relationship between two fluorescent dye signals for at least 20 individual cells ± SE. The colors of the representative image correspond to the drawing outline.

Example 3

CD 8T cell fatigue biomarker signature

TBET, EOMES and PD1 may define the fatigue or effector biomarker signature of T cells. Fatigue biomarker signatures comprise TBET-low, EOMES-high, PD 1-high, while effect biomarker signatures comprise TBET-high, EOMES-low, PD 1-low.

The present inventors examined the expression of these markers in the CR, SD and PD groups and found that: 1) EOMES and PD1 were highly expressed in the PD group, whereas TBET was significantly lower than the CR/SD group; 2) TBET is highly expressed in the CR/SD group and is less expressed in PD; 3) PKC θ i targets the fatigue pathway, inhibits expression of EOMES and PD-1, and other T cell activation pathways. This allows PKC-theta inhibition to simultaneously inhibit the exhausted pathway and enhance TBET expression; 4) this allows PKC θ i to epigenetically reprogram T cells from an exhausted biomarker signature to an effector/active T cell biomarker signature; and 5) inhibition of PD-1 by PKC-theta inhibitors (e.g., PKC theta i) suggests that this would further aid PD-1 immunotherapy.

Method of producing a composite material

Using RosetteSep^TMMethod for pre-enriching metastatic melanoma biopsy tissue using RosetteSep^TMHuman CD8 enrichment kit (15063, StemCell Technologies) for CTC isolation and SepMate^TM-50(IVD) density gradient tubes (85450, StemShell Technologies) and Lymphoprep^TMDensity gradient media (07861, stemcell technologies) were subjected to density gradient centrifugation to separate CD8+ cells from red blood cells. Enriched CTC cells were then stimulated with vehicle or PMA/CI and preclinical screening was performed with 8mm concentration controls or our proprietary novel nuclear PKC-theta inhibitors. The enriched cells were then centrifuged, smeared onto coverslips pretreated with poly-1-lysine and fixed, and then stored in PBS for staining. To examine melanoma CD8 treated with PKC θ i⁺Kinetics of EOMES, TBET and PD-1 in T cells, CD8⁺T cells were incubated with 1% Triton X-100 for 20 minutes to permeabilize them and probed with rabbit anti-PKC-theta (T538p), mouse anti-ZEB 1 and goat anti-CD 8 or IFN- γ (mouse), TNF- α (rabbit) and goat anti-CD 8 and visualized by donkey anti-rabbit AF488, anti-mouse 568 and anti-goat hi mounting a coverslip on a glass microscope slide with pro long Diamond anti reagent (life technologies) protein targets were located by confocal laser scanning microscopy.

The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

Citation of any reference herein shall not be construed as an admission that such reference is available as "prior art" to the present application.

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Thus, those skilled in the art will appreciate that, in light of the present disclosure, various modifications and changes may be made in the specific embodiments illustrated without departing from the scope of the present invention. All such modifications and variations are intended to be included herein within the scope of the appended claims.

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Claims (96)

1. A composition for enhancing T cells (e.g., CD 8)⁺T cell) function, or for use in treating a T cell dysfunctional disorder, the composition comprising, or consisting essentially of, an inhibitor of PKC-theta and a PD-1 binding antagonist.

2. The composition according to claim 1, wherein the PKC-theta inhibitor is an inhibitor of PKC-theta nuclear translocation.

3. The composition according to claim 2, wherein the inhibitor of PKC- Θ is a peptide corresponding to a nuclear localization site of PKC- Θ.

4. The composition of claim 3, wherein the PKC-theta inhibitor is a protein molecule represented by formula (XXVI):

Z₁X₁X₂X₃X₄IDX₅PPX₆X₇X₈X₉X₁₀X₁₁Z₂(XXVI)

wherein:

"Z1" and "Z2" are independently absent or independently selected from at least one of a protein moiety and a protecting moiety; the protein moiety comprises from about 1 to about 50 amino acid residues (and all integer amino acid residues therebetween);

“X₁"absent or selected from basic amino acid residues including R, K and modified forms thereof;

“X₂"and" X₃"is independently selected from the group consisting of basic amino acid residues including R, K and modified forms thereof;

“X₄"is selected from charged amino acid residues including R, K, D, E and modified forms thereof;

“X₅"absent or as W or modified form thereof;

“X₆"is selected from aromatic or basic amino acid residues including F, Y, W, R, K and modified forms thereof;

“X₇"is selected from the group consisting of basic amino acid residues including R, K and modified forms thereof;

“X₈"absent or is P or modified form thereof;

“X₉"is selected from the group consisting of basic amino acid residues including R, K and modified forms thereof;

“X₁₀"is selected from the group consisting of hydrophobic residues including V, L, I, M and modified forms thereof and P and modified forms thereof;

“X₁₁"is selected from the group consisting of basic amino acid residues, including R, K and modified forms thereof.

5. The composition of claim 4, wherein "X" is₁"to" X₁₁"selected from the group consisting of one or more of:

“X₁"absent or R;

“X₂"is R;

“X₃"is K;

“X₄"is E or R;

“X₅"absent or is W;

“X₆"is F or R;

“X₇"is R;

“X₈"absent or is P;

“X₉"is K;

“X₁₀"is V or P; and

“X₁₁"is K.

6. The composition of claim 4 or 5, wherein "Z" is₁"consists of 10, 9, 8, 7,6, 5, 4, 3, 2 or1 amino acid residue.

7. The composition according to any one of claims 4 to 6, wherein "Z" is₂"consists of 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 amino acid residue.

8. The composition according to any one of claims 4 to 7, wherein "Z" is₁"and" Z₂The amino acid residue in "is selected from any amino acid residue.

9. The composition of claim 4 or 5, wherein "Z" is₁"is a protein molecule represented by formula XXVII:

X₁₂X₁₃X₁₄X₁₅X₁₆(XXVII)

wherein

“X₁₂"absent or a protected moiety;

“X₁₃"absent or selected from P and basic amino acid residues, wherein said basic amino acid residues include R, K and modified forms thereof;

“X₁₄"absent or selected from P and basic amino acid residues, wherein said basic amino acid residues include R, K and modified forms thereof;

“X₁₅"absent or selected from P and basic amino acid residues, wherein said basic amino acid residues include R, K and modified forms thereof;

“X₁₆"absent or selected from P and basic amino acid residues, wherein said basic amino acid residues include R, K and modified forms thereof.

10. The composition of any one of claims 4,5 and 9, wherein "Z" is₂"is a protein molecule represented by formula XXVIII:

X₁₇X₁₈X₁₉X₂₀(XXVIII)

wherein:

“X₁₇"absent or selected from any amino acid residue;

“X₁₈"absent or selected from any amino acid residue;

“X₁₉"absent or selected from any amino acid residue;

“X₂₀"absent or a protected moiety.

11. The composition of claim 4 or 5, wherein "Z" is₁"and" Z₂"absent.

12. The composition of claim 4, wherein the protein molecule of formula XXVI comprises the amino acid sequence of SEQ ID NO: 4 or 5, consisting of the amino acid sequence represented by SEQ ID NO: 4 or 5, or consists essentially of the amino acid sequence represented by SEQ ID NO: 4 or 5, and the amino acid sequence represented by SEQ ID NO: 4 or 5 are as follows:

RKEIDPPFRPKVK [SEQ ID NO：4]

RRKRIDWPPRRKPK [SEQ ID NO：5]。

13. the composition according to claim 1, wherein the PKC- Θ inhibitor is an inhibitor of PKC- Θ enzyme activity.

14. The composition of any one of claims 1 to 13, wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2.

15. The composition of any one of claims 1 to 14, wherein the PD-1 binding antagonist is an anti-PD-1 antagonist antibody.

16. The composition of claim 15, wherein the anti-PD-1 antagonist antibody is selected from nivolumab, pembrolizumab, lambertizumab, and pidilizumab.

17. The composition of any one of claims 1-14, wherein the PD-1 binding antagonist is an immunoadhesin (e.g., AMP-224).

18. The composition of any one of claims 1 to 17, further comprising an adjuvant (e.g., a chemotherapeutic agent) for use in the treatment or for use in the adjunctive treatment of a T cell dysfunctional disorder.

19. The composition of any one of claims 1 to 18, further comprising a pharmaceutically acceptable carrier.

20. A method of enhancing T cell function, the method comprising, consisting of, or consisting essentially of: contacting a T cell with a PKC-theta inhibitor and a PD-1 binding antagonist, thereby enhancing T cell function.

21. The method of claim 20, wherein enhancing T cell function comprises any one or more of increasing production of cytokines such as IL-2, IFN- γ, TNF- α, increasing CD8⁺Activation of T cells, increased recognition by T cell receptors of antigens or antigenic peptides derived from MHC class I molecules, increased clearance of cells presented by MHC class I molecules, and increased cytolytic killing of target cells expressing the antigen.

22. The method of claim 20 or 21, wherein the T cell has a mesenchymal phenotype.

23. The method of any one of claims 20-22, wherein the T cell has aberrant expression of nuclear PKC- Θ.

24. The method of claim 23, wherein the T cell expresses nuclear PKC-theta at a level greater than the level of expression of TBET in the same T cell, and/or the T cell expresses nuclear PKC-theta at a level greater than the level of expression in an activated T cell.

25. The method of any one of claims 20 to 24, wherein said T cell is a T cell that exhibits T cell fatigue or anergy.

26. The method of claim 25, wherein the T cell expresses EOMES at a level greater than the level of TBET and/or has increased PD-1 expression.

27. The method of any one of claims 20-26, wherein the T cell is CD8⁺T cells.

28. A method of enhancing an immune effector function of an immune effector cell expressing PD-1, the method comprising, consisting of, or consisting essentially of: contacting an immune effector cell with a PKC-theta inhibitor and a PD-1 binding antagonist, thereby enhancing an immune effector function of the immune effector cell.

29. The method of claim 28, wherein the enhanced immune effector function comprises any one or more of: increasing recognition by T cell receptors of antigens or antigenic peptides derived from MHC class II molecules, increasing cytokine release and/or CD8⁺Activation of lymphocytes (CTL) and/or B cells, increased recognition by T cell receptors of antigens or antigenic peptides derived from MHC class I molecules, increased clearance of cells presented by MHC class I molecules, i.e., cells characterized by presentation of antigens by MHC class I, e.g., by apoptosis or perforin-mediated cell lysis, increased production of cytokines such as IL-2, IFN- γ, TNF- α, and increased specific cytolytic killing of antigen-expressing target cells.

30. The method of claim 29, wherein the immune effector cell expresses a level of nuclear PKC- Θ that is greater than the level of expression of a control immune effector cell (e.g., an immune effector cell having normal or non-suppressed immune effector function).

31. A method of treating a T cell dysfunctional disorder in a subject, comprising, consisting of, or consisting essentially of: administering to the subject an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist concurrently to treat a T cell dysfunctional disorder.

32. The method of claim 31, wherein the PKC- Θ inhibitor and the PD-1 binding antagonist are administered in synergistically effective amounts.

33. The method of claim 31 or 32, wherein the T cell dysfunctional disorder is a disorder or condition of T cells characterized by a decreased response to antigen stimulation and/or increased inhibitory signal transduction by PD-1.

34. The method of any one of claims 31-33, wherein the T cell dysfunctional disorder is a disorder in which T cells have reduced ability to secrete cytokines, proliferate, or undergo cytolytic activity.

35. The method of any one of claims 31 to 34, wherein a reduced response to antigen stimulation results in ineffective control of a pathogen or tumor.

36. The method of any one of claims 31-35, wherein the T cell dysfunctional disorder is one in which T cells are anergic.

37. The method of any one of claims 31-36, wherein the T cell dysfunctional disorder is selected from the group consisting of unresolved acute infection, chronic infection, and tumor immunity.

38. The method of any one of claims 31-37, wherein the T cell dysfunctional disorder is a cancer or infection comprising T cells having a mesenchymal phenotype (e.g., CD 8)⁺T is thinA cell).

39. The method of any one of claims 31-38, wherein the T cell expresses nuclear PKC-theta at a level greater than the level of expression of TBET in the same T cell, and/or the T cell expresses nuclear PKC-theta at a level greater than the level of expression in an activated T cell.

40. The method of any one of claims 31-39, wherein said T cell is a T cell that exhibits T cell fatigue or anergy.

41. The method of any one of claims 31 to 40, wherein the T cells express EOMES at a level greater than the level of TBET and/or have increased PD-1 expression.

42. The method of any one of claims 31-41, wherein the T cells are tumor infiltrating lymphocytes.

43. The method of any one of claims 31-41, wherein the T cell is a circulating lymphocyte.

44. The method of any one of claims 31-43, wherein the cancer is skin cancer (e.g., melanoma), lung cancer, breast cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, renal cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, or hepatocellular carcinoma.

45. The method of claim 44, wherein the cancer is a metastatic cancer.

46. The method of claim 45, wherein the metastatic cancer is metastatic melanoma or metastatic lung cancer.

47. The method of any one of claims 31 to 43, further comprising simultaneously administering to the subject a PKC-theta inhibitor and a PD-1 binding antagonist, adjuvant (e.g., chemotherapeutic agent), or adjuvant therapy (e.g., ablative or cytotoxic therapy) for the treatment or adjuvant treatment of a T cell dysfunctional disorder.

48. A method of treating or delaying progression of cancer in a subject, the method comprising, consisting of, or consisting essentially of: administering to the subject an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist concurrently to treat or delay progression of the cancer.

49. The method of claim 48, wherein the subject has been diagnosed with cancer, wherein in a tumor sample of the cancer from the subject, the T cells express nuclear PKC-theta at a level greater than the expression level of TBET in the same T cells, and/or the T cells express nuclear PKC-theta at a level greater than the expression level in activated T cells.

50. A method of enhancing immune function (e.g., immune effector function) in an individual having cancer, the method comprising, consisting of, or consisting essentially of: administering to the individual an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist simultaneously to enhance immune function.

51. The method of claim 50, wherein the individual has been diagnosed with cancer, wherein a tumor sample of the cancer taken from the individual has a level of T cells expressing nuclear PKC- θ that is greater than the level of expression of TBET in the same T cells, and/or greater than the level of expression in activated T cells.

52. A method of treating an infection (e.g., a bacterial or viral or other pathogen infection), the method comprising, consisting of, or consisting essentially of: administering to the individual an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist simultaneously to treat the infection.

53. The method of claim 52, wherein the infection is a viral and/or bacterial infection.

54. The method of claim 52, wherein the infection is a pathogen infection.

55. The method of any one of claims 52-54, wherein the infection is an acute infection.

56. The method of any one of claims 52-54, wherein the infection is a chronic infection.

57. A method of enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having an infection, the method comprising, consisting of, or consisting essentially of: administering to the individual an effective amount of a PKC-theta inhibitor and a PD-1 binding antagonist simultaneously to enhance immune function.

58. The method of claim 57, wherein the individual has been diagnosed with an infection, wherein in the sample from the individual, the T cells express nuclear PKC- θ at a level that is greater than the level of expression of TBET in the same T cells, and/or greater than the level of expression in activated T cells.

Use of a PKC-theta inhibitor and a PD-1 binding antagonist for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection.

Use of a PKC-theta inhibitor and a PD-1 binding antagonist in the manufacture of a medicament for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection.

61. The use of claim 59 or 60, wherein the PKC-theta inhibitor and PD-1 binding antagonist are formulated for simultaneous administration.

Use of PKC-theta inhibitors, PD-1 binding antagonists and adjunctive agents (e.g., chemotherapeutic agents) for treatment or for adjunctive treatment of T cell dysfunctional disorders, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, for treating or delaying progression of cancer, or for treating infection.

63. PKC-theta inhibitors, PD-1 binding antagonists and adjuvants (e.g., chemotherapeutic agents) for the manufacture of medicaments for the treatment or for the adjunctive treatment of T cell dysfunctional disorders, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in individuals with cancer, for treating or delaying the progression of cancer, or for treating infections.

64. The use of claim 62 or 63, wherein the PKC-theta inhibitor, PD-1 binding antagonist, and adjuvant (e.g., chemotherapeutic agent) are formulated for simultaneous administration.

65. The method of any one of claims 31-58, further comprising, prior to the concurrently administering, detecting elevated nuclear PKC-theta (i.e., PKC-theta localized in the nucleus) levels (e.g., relative to TBET levels in the same T cell, or nuclear PKC-theta levels in an activated T cell) in T cells in a sample obtained from the subject.

66. The method of any one of claims 31 to 58, further comprising, prior to the concurrently administering, detecting elevated nuclear PKC-theta (i.e., PKC-theta localized in the nucleus) levels (e.g., relative to TBET levels in the same T cell, or nuclear PKC-theta levels in an activated T cell) in T cells in a sample obtained from the subject, and elevated ZEB1 levels in the nucleus of the T cells (e.g., relative to TBET levels in the same T cell, or the levels of ZEB1 in the nucleus of the activated T cell).

67. The method of claim 66, comprising detecting an elevated level of a complex comprising PKC- θ and ZEB 1.

68. The method of claim 66, comprising detecting an elevated level of a complex comprising PKC-theta and ZEB1 in the nucleus of a T cell.

69. A kit comprising a medicament comprising a PKC-theta inhibitor and optionally a pharmaceutically acceptable carrier, and a package insert comprising instructional material for simultaneously administering the medicament and another medicament comprising a PD-1 binding antagonist and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying the progression of cancer, or for treating an infection in an individual.

70. A kit comprising a medicament comprising a PD-1 binding antagonist and optionally a pharmaceutically acceptable carrier, and a package insert comprising instructional material for simultaneously administering the medicament and another medicament comprising a PKC-theta inhibitor and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying the progression of cancer, or for treating an infection in an individual.

71. A kit comprising a first medicament comprising a PKC-theta inhibitor and optionally a pharmaceutically acceptable carrier, and a second medicament comprising a PD-1 binding antagonist and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection in an individual.

72. The kit of claim 71, further comprising a package insert comprising instructional material for simultaneously administering the first drug and the second drug for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual having cancer, for treating or delaying progression of cancer, or for treating an infection in an individual.

73. The method of any one of claims 31-66, wherein CD8 in the individual compared to prior to administration of the combination⁺T cells have enhanced priming, activation, proliferation and/or cytolytic activity.

74. The method of any one of claims 31-66 and 73, wherein CD8 is compared to before administration of the combination⁺The number of T cells increases.

75. The method of claim 74, wherein the CD8⁺T cells are antigen-specific CD8⁺T cells.

76. The method according to any one of claims 31 to 66 and 73 to 75, wherein Treg function is inhibited compared to before administration of the combination of the PKC-theta inhibitor and the PD-1 binding antagonist.

77. The method of any one of claims 31-66 and 73-76, wherein T cell fatigue is reduced as compared to prior to administration of the combination of the PKC- Θ inhibitor and the PD-1 binding antagonist.

78. The method of any one of claims 31-66 and 73-77, wherein the number of Treg cells is reduced as compared to before administration of the combination of the PKC-theta inhibitor and the PD-1 binding antagonist.

79. The method of any one of claims 31-66 and 73-78, wherein plasma IFN- γ is increased as compared to before administration of the combination of the PKC-theta inhibitor and the PD-1 binding antagonist.

80. The method of any one of claims 31-66 and 73-79, wherein plasma TNF-a is increased as compared to before the administration of the combination of the PKC-theta inhibitor and the PD-1 binding antagonist.

81. The method of any one of claims 31-66 and 73-80, wherein plasma IL-2 is increased as compared to before administration of the combination of the PKC- Θ inhibitor and the PD-1 binding antagonist.

82. The method of any one of claims 31-66 and 73-81, wherein the number of memory T effector cells is increased as compared to before administration of the combination of the PKC-theta inhibitor and the PD-1 binding antagonist.

83. The method of any one of claims 31-66 and 73-82, wherein activation and/or proliferation of memory T effector cells is increased as compared to before administration of the combination of a PKC-theta inhibitor and a PD-1 binding antagonist.

84. The method of any one of claims 31-66 and 73-83, wherein memory T effector cells are detected in peripheral blood.

85. The method of claim 84, wherein memory T effector cells are detected by detecting CXCR 3.

86. A method of diagnosing the presence of a T cell dysfunctional disorder in a subject, comprising, consisting or consisting essentially of the steps of:

(i) obtaining a sample from a subject, wherein the sample comprises T cells (e.g., CD 8)⁺T cells);

(ii) contacting the sample with a first binding agent that binds PKC-theta in the sample and a second binding agent that binds ZEB1 in the sample; and

(iii) detecting the localization of the first and second binding reagents in the nucleus of the T cell;

wherein localization of the first binding agent and the second binding agent in the nucleus of the T cell is indicative of the presence of a T cell dysfunctional disorder in the subject.

87. In yet another aspect, the invention provides a method of diagnosing the presence of a T cell dysfunctional disorder in a subject, comprising, consisting or consisting essentially of the steps of:

(i) obtaining a sample from a subject, wherein the sample comprises T cells (e.g., CD 8)⁺T cells);

(ii) contacting the sample with a first binding agent that binds PKC-theta in the sample and a second binding agent that binds ZEB1 in the sample; and

(iii) detecting the first binding reagent and the second binding reagent when bound to PKC-theta-ZEB 1 complex in the sample;

wherein an increase in the level of PKC-theta-ZEB 1 complex detected in the sample relative to the level of PKC-theta-ZEB 1 complex detected in a control sample (e.g., a sample comprising activated T cells) is indicative of the presence of a T cell dysfunctional disorder in the subject.

88. A method of monitoring treatment of a subject having a T cell dysfunctional disorder, comprising, consisting or consisting essentially of the steps of:

(i) obtaining a sample from a subject following treatment of the subject with a therapy for a T cell dysfunctional disorder, wherein the sample comprises T cells (e.g., CD 8)⁺T cells);

(ii) contacting the sample with a first binding agent that binds PKC-theta in the sample and a second binding agent that binds ZEB1 in the sample; and

(iii) detecting the first binding agent and the second binding agent when bound to PKC-theta-ZEB 1 complex in the sample;

wherein a lower level of PKC-theta-ZEB 1 complex detected in the sample relative to the level of PKC-theta-ZEB 1 complex detected in a control sample obtained from the subject prior to treatment indicates an increased clinical benefit (e.g., enhanced immune effector function, e.g., enhanced T cell function) in the subject, and

wherein a higher level of PKC-theta-ZEB 1 complex detected in the sample relative to the level of PKC-theta-ZEB 1 complex detected in a control sample obtained from the subject prior to treatment indicates no or negligible clinical benefit to the subject (e.g., enhanced immune effector function, e.g., enhanced T cell function).

89. A kit for diagnosing the presence of a T cell dysfunctional disorder in a subject, the kit generally comprising, consisting of, or consisting essentially of: (i) a first binding agent that binds to PKC-theta, (ii) a second binding agent that binds to ZEB 1; and (iii) a third agent comprising a label that is detectable when the first binding agent and the second binding agent each bind to a PKC-theta-ZEB 1 complex.

90. The kit of claim 89, wherein the third reagent is a binding reagent that binds to the first and second binding reagents.

91. A complex comprising PKC-theta and ZEB1, a first binding agent that binds PKC-theta of said complex, a second binding agent that binds ZEB1 of said complex; and (iii) a third agent comprising a label that is detectable when the first binding agent and the second binding agent each bind to a PKC-theta-ZEB 1 complex.

92. The complex of claim 91, wherein the PKC- Θ -ZEB1 complex is localized in a T cell.

93. The complex of claim 91 or claim 92, wherein the third agent is a binding agent that binds to the first and second binding agents.

94. A T cell comprising a complex comprising PKC-theta and ZEB1, a first binding agent that binds to PKC-theta of the complex, a second binding agent that binds to ZEB1 of the complex; and (iii) a third agent comprising a label that is detectable when the first binding agent and the second binding agent each bind to a PKC-theta-ZEB 1 complex.

95. The T cell of claim 94, wherein the third agent is a binding agent that binds to the first and second binding agents.

96. The method, kit, complex or T cell of claims 86-95, wherein the corresponding binding reagent is an antibody.

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